Nanocluster compositions and methods

ABSTRACT

Compositions, methods of making, and methods of using nanoclusters in which the nanoclusters comprise a plurality of nanoparticles having a core of nanoparticles arranged such that the surfaces of the nanoparticles contact adjacent nanoparticles, the nanoparticles comprise an active ingredient, and the nanocluster has a mass median aerodynamic diameter of from about 0.25 μm to about 20 μm.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/US/09/50565, entitled“Nanoclusters for Delivery of Poorly Water Soluble Drug Nanoparticles,”filed on Jul. 14, 2009, which claims the benefit of Provisionalapplication Nos. 61/081,034, filed on Jul. 15, 2008 and 61/081,037,filed on Jul. 16, 2008, and also a continuation-in-part of U.S. patentapplication Ser. No. 12/261,907, entitled “Nanoclusters for Delivery ofTherapeutics,” filed on Oct. 30, 2008 now abandoned, and also acontinuation-in-part of U.S. patent application Ser. No. 12/261,873,entitled “Nanoclusters for Delivery of Therapeutics,” filed on Oct. 30,2008 now abandoned, both of which are divisionals of U.S. patentapplication Ser. No. 11/610,986, filed on Dec. 14, 2006 now U.S. Pat.No. 7,651,770, which claims the benefit of Provisional application No.60/751,172 filed Dec. 16, 2005, all of which are incorporated byreference.

BACKGROUND

Millions of people worldwide suffer from a wide variety of diseases orconditions that would benefit from the effective delivery of therapeuticor diagnostic agents. Examples of these diseases or conditions includepulmonary diseases, circulatory diseases, muscular diseases, bonediseases, and cancers.

Current drug delivery treatment options can often be ineffective due toinefficient delivery of an active ingredient to a targeted site. The useof nanoparticles as drug delivery vehicles has been employed for avariety of indications. Nanoparticles, for example, have been shown toimprove the dissolution of poorly water-soluble drugs and enhance thetransport of drugs both intra- and paracellularly. In addition,literature indicates that plasmid DNA can be effectively delivered bypolycationic polymers that form nanoparticles when mixed with DNAresulting in enhanced gene expression. Although nanoparticles offerseveral advantages for delivering drugs (e.g. improved dissolution oflow solubility API, intracellular and transcellular transport, etc.),the use of nanoparticles, for example, can be hindered by the inabilityto deliver nanoparticles to the site of drug action (e.g. driednanoparticles are too small to deposit efficiently in the lungs, canavoid detection by APCs, etc.). In addition, nanoparticles are oftendifficult to handle at an industrial scale.

SUMMARY

The present disclosure relates to the delivery of therapeutic anddiagnostic agents, and more particularly, in certain embodiments, to ananocluster drug delivery platform.

The present disclosure provides, according to certain embodiments,nanoclusters comprising a plurality of nanoparticles having a core ofnanoparticles arranged such that the surfaces of the nanoparticlescontact adjacent nanoparticles, the nanoparticles comprise activeingredient, and the nanocluster has a mass median aerodynamic diameterof from about 0.25 μm to about 20 μm.

The present disclosure provides, according to certain embodiments,methods comprising forming a nanocluster comprised of a core ofnanoparticles arranged such that the surface of the nanoparticlescontact one another, the nanoparticles comprise an active ingredient,wherein the nanocluster has a mass median aerodynamic diameter of fromabout 0.25 μm to about 20 μM.

The present disclosure provides, according to certain embodiments,methods comprising administering to a subject a nanocluster comprised ofa core of nanoparticles arranged such that the surface of thenanoparticles contact one another, the nanoparticles comprise an activeingredient, wherein the nanocluster has a mass median aerodynamicdiameter of from about 0.25 μm to about 20 μm.

The present disclosure provides, according to certain embodiments,compositions comprising a nanocluster, the nanocluster comprises aplurality of nanoparticles having a core of nanoparticles arranged suchthat the surfaces of the nanoparticles contact adjacent nanoparticles,the nanoparticles comprise an active ingredient, and the nanocluster hasa mass median aerodynamic diameter of from about 0.25 μm to about 20 μm.

The features and advantages of the present disclosure will be readilyapparent to those skilled in the art upon a reading of the descriptionof the embodiments that follows

DRAWINGS

Some specific example embodiments of the disclosure may be understood byreferring, in part, to the following description and the accompanyingdrawings.

FIG. 1. Uniform (˜75 μm) nanoclusters composed of polystyrenenanoparticles.

FIG. 2. Electron micrographs of (A) 225 nm silica nanoparticles coatedwith a dispersion material (light gray corona) and (B) a 9 μmnanocluster of the silica nanoparticles coated with dispersion material.

FIG. 3. The dispersion of nanoclusters over time composed ofnanoparticles coated with a hydrolysable polymer was a function of pH asdetermined by (A) absorption of light at 480 nm and (B) visualinspection. (C) Size analysis of the dispersion shows polydisperseagglomerates are liberated from the nanoclusters, which then break downinto monodisperse nanoparticles.

FIG. 4A, FIG. 4B, FIG. 4C. The (FIG. 4A) geometric and (FIG. 4B)aerodynamic size distributions of PLGA nanoclusters produced byincreasing the concentration of nanoparticles (black=0.68 mg/ml,red=1.36 mg/ml, green=2.16 mg/ml, blue=2.72 mg/ml). FIG. 4C Scanningelectron micrograph of nanocluster structure. Scale bar=5 μm.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F. Laser scanningconfocal micrographs of PLGA nanoparticle nanoclusters. FITC-labeledPVAm-coated nanoparticles (FIG. 5A and FIG. 5D) and rhodamine-labeledPEMA-coated nanoparticles (FIG. 5B and FIG. 5E) are both identifiedwithin the nanocluster structure. FIG. 5C and FIG. 5F Overlays of themicrographs reveal the diffuse structure of the nanoclusters. Scalebar=5 μm.

FIG. 6. Scanning electron microscope (SEM) image of a population ofnifedipine nanoparticles.

FIG. 7. SEM image of nifedipine nanoparticle clusters.

FIG. 8 Illustration of the geometric diameter of the nanoclusterscomprising DOTAP/PLGA nanoparticles and ovalbumin.

FIG. 9. SEM images of the nanoclusters comprising DOTAP/PLGAnanoparticles and ovalbumin.

FIG. 10. The particle size distributions of paclitaxel nanoparticleagglomerates in suspension after agglomeration and resuspended afterlyophilization.

FIG. 11. Aerodynamic size distributions of paclitaxel nanoparticleagglomerates after lyophilization.

FIG. 12. The distribution of paclitaxel powder as received andnanoparticle agglomerate formulations deposited on the stages of acascade impactor at a flow rate of ˜30 L/min.

FIG. 13. In-vitro dissolution profiles of paclitaxel in PBS (pH 7.4)from pure paclitaxel powder and two different nanoparticle (NP) andnanoparticle agglomerate formulations (NA).

FIG. 14. Viability of A549 cells in the presence of formulationcomponents as determined by an MTS assay.

FIG. 15. The particle size distributions of budesonide nanoparticleagglomerates in suspension after agglomeration and resuspended afterlyophilization.

FIG. 16. Aerodynamic size distributions of budesonide nanoparticleagglomerates after lyophilization.

FIG. 17. The distribution of budesonide nanoparticle agglomerateformulations (A) F1, (B) F2, and (C) F3 deposited on the stages of acascade impactor. (D) Formulations were compared with stock budesonideat a flow rate of ˜30 L/min.

FIG. 18. Transmission electron micrographs of A) F1 nanoparticles and B)F1 nanoparticle agglomerates.

FIG. 19. 13C CP/MAS spectra of budesonide, excipients, and budesonideformulations. The nanoparticle agglomerates spectrum was expanded 8times vertically to produce the nanoparticle agglomerates ×8 spectrum toaid the interpretation of the budesonide peaks.

FIG. 20. Structure of budesonide with carbon numbering.

FIG. 21. 13C CP/MAS spectra from spectral editing experiment. All=allcarbon types are shown, C+CH3=only unprotonated and methyl carbons areshown, C=only unprotonated carbons are shown, CH=only methine carbonsare shown, and CH2=only methylene carbons are shown.

FIG. 22. In-vitro dissolution profiles of budesonide in PBS (pH 7.4)from budesonide stock and three different nanoparticle (NP) andnanoparticle agglomerate formulations (NA).

FIG. 23. Viability of A549 cells in the presence of formulationcomponents as determined by an MTS assay. Bud: budesonide; Lec: Lecithinstock; Leu: Leucine stock; PM: Physical mixture of F1 components(0.1:0.02:0.1; Bud:Lec:Leu); B1. NA: F1 Blank nanoparticle agglomerates;NP: F1 nanoparticles; NA: F1 nanoparticle agglomerates.

FIG. 24. Percent volume as a function of particle diameter for aagglomerated solution of NIF/SA nanoparticles in water (421.7+/−26.2 nm,−32.16+/−3.75 mV) after addition of NaCl to 0.1M. Also shown is the samesolution after homogenization at 25000 RPM for 30 seconds.

FIG. 25. Aerodynamic Diameter size distribution for the sample ofnanoparticle agglomerates.

FIG. 26. A collection of SEM images for nanoparticles directly aftersonication (A), newly prepared agglomerates (B), agglomerate powdersafter residing under room conditions and devoid of light for 1 month(C), and pure nifedipine crystals as received (D).

FIG. 27. DSC outputs for the optimal formulation of nanoparticles, purenifedipine, and agglomerated nanoparticles.

FIG. 28. Percent drug dissolution vs. Time as deduced via HPLC UVspectroscopy for the nifedipine/stearic acid nanoparticles,agglomerates, and the drug in pure crystalline form.

FIG. 29. Cascade impactor readings for nifedipine/stearic acidnanoparticles, agglomerates, and drug as received in pure form.

FIG. 30. Outline of insulin processing method.

FIG. 31. Mass fraction of insulin in pellet vs. PH. Each valuerepresents mean±standard deviation of three experiments.

FIG. 32. Nanocluster size vs. Nanoparticle size. Each value representsmean±standard deviation of three experiments.

FIG. 33. SEM micrographs of insulin particles; (A) and (B) areunprocessed insulin particles (scale bars 30 pm and 10 pm,respectively); (C) and (D) are insulin nanoclusters after processing(scale bars 10 pm and 2 pm, respectively).

FIG. 34. Tap density of insulin powders. Each bar shows mean±standarddeviation of three experiments.

FIG. 35. Circular dichroism of dissolved insulin powders. The top panelshows isothermal spectra, and the bottom panel shows variabletemperature scan at a wavelength of 210 nm. Each value of the variabletemperature scan represents mean±standard deviation of threeexperiments.

FIG. 36. 13C CP/MAS NMR spectra for insulin powders; (A) Unprocessed;(B)

Insulin nanoclusters; (C) Lyophilized insulin nanoparticles; (D)Centrifuged and dried insulin nanoparticles.

FIG. 37. Percent crystallinity of insulin particles, as determined bythe HPLC dissolution method described in the U.S. Pharmacopeia andNational Formulary. Each bar shows mean±standard deviation of threeexperiments.

FIG. 38. Dissolution of insulin powders over time. Each value representsmean±standard deviation of three experiments.

FIG. 39. The distribution of diatrizoic acid powder as received as wellas nanoparticle agglomerate. D9 S: Resuspended Diatrizoic acidnanoparticle agglomerates; D9 P: Diatriazoic acid nanoparticleagglomerated dry powder; Dia S: Resuspended Diatrizoic acid powder asreceived; Dia P: Diatrizoic acid powder as received.

FIG. 40. Size distribution of moxifloxacin nanoparticles suspension M5.

FIG. 41. The particle size distributions of Ciprofloxacin nanoparticleagglomerates C1 after flocculation and resuspended after lyophilization.

FIG. 42. Aerodynamic size distributions of Ciprofloxacin nanoparticleagglomerates C1 after lyophilization.

FIG. 43. The distribution of Ciprofloxacin powder as received as well asnanoparticle agglomerate formulation (C1) deposited on the stages of acascade impactor at a flow rate of ˜30 L/min.

FIG. 44A/B. Transmission electron micrographs of diatrizoic acid A) D1nanoparticles and B) D1 nanoparticle agglomerates.

FIG. 45. Dissolution profiles of Ciprofloxacin in PBS (pH 7.4) fromCiprofloxacin powder as received as well as the prepared C1 nanoparticle(NP) and nanoparticle agglomerate formulation (NA).

FIG. 46. Rifampicin as received.

FIG. 47. Rifampicin/acetone/water system.

FIG. 48. Rifampicin/ethanol/water system.

FIG. 49. Drug as received.

FIG. 50. SEM of SV 4-7.

FIG. 51. SEM of SV 5-7.

FIG. 52. SEM of SV 3-3.5.

FIG. 53. SEM of SV 4-3.5.

FIG. 54. SEM of SV 2-3.5.

FIG. 55. SEM of LV 2-3.5.

FIG. 56. SEM of LV 2-3.5/1% Lecithin.

FIG. 57. SEM of LV 2-3.5/0.1% oleic acid.

FIG. 58. SEM of SV 2-3.5/0.3% L-leucine.

FIG. 59. SEM of SV 2-3.5/0.7% L-leucine.

FIG. 60. SEM of SV 2-3.5/1% L-leucine.

FIG. 61. SEM of SV 2-3.5/0.3% Lactose.

FIG. 62. SEM of SV 2-3.5/0.7% Lactose.

FIG. 63. SEM of SV 2-3.5/1% Lactose.

FIG. 64. The aerodynamic size distribution of budesonide NanoClusterpowder of Batch 2 (200 mic), milled for 3 hours, at a rate of 30 L/minusing Spinhaler and low feed.

FIG. 65. The aerodynamic size distribution of budesonide NanoClusterpowder of Batch 3 (200 mic), milled for 8 hours, at a rate of 30 L/minusing Spinhaler and low feed.

FIG. 66. SEM of milling with 200 micron media for 5 hrs.

FIG. 67. SEM of milling with 200 micron media for 5.5 hrs.

FIG. 68. SEM of milling with 200 micron media for 6 hrs.

FIG. 69. SEM of milling with 200 micron media for 6.5 hrs.

FIG. 70. SEM of milling with 200 micron media for 7 hrs.

FIG. 71. SEM of milling with 200 micron media for 7.5 hrs.

FIG. 72. SEM of milling with 200 micron media for 8 hrs.

FIG. 73. HPLC assay of 200 micron media batch 3 after 8 hrs milling.

FIG. 74. Focusing on the degradation peak of the samples.

FIG. 75. HPLC assay of 200 micron media batch 4 (the name methanol issample at 10 hrs).

FIG. 76. Focus on the degradation peaks of 200 micron media batch 4 (thename methanol is sample at 10 hrs).

FIG. 77. Milling with 200 micron media for 9 hrs.

FIG. 78. Milling with 200 micron media for 10 hrs.

FIG. 79. Milling with 200 micron media for 10 hrs/0.3% w/w lactose.

FIG. 80. Milling with 200 micron media for 10 hrs/1% w/w lactose.

FIG. 81. Milling with 200 micron media for 10 hrs/0.3% w/w leucine.

FIG. 82. Milling with 200 micron media for 10 hrs/1% w/w leucine.

FIG. 83. Stability results for Concentration: 0.4 mg/ml at Time: 0 min.

FIG. 84. Stability results for Concentration: 0.4 mg/ml at Time: 1 hr.

FIG. 85. Stability results for Concentration: 0.4 mg/ml at Time: 4 days.

FIG. 86. Stability results for Concentration: 0.2 mg/ml at Time: 0 min.

FIG. 87. Stability results for Concentration: 0.2 mg/ml at Time: 3 days.

While the present disclosure is susceptible to various modifications andalternative forms, specific example embodiments have been shown in thefigures and are herein described in more detail. It should beunderstood, however, that the description of specific exampleembodiments is not intended to limit the invention to the particularforms disclosed, but on the contrary, this disclosure is to cover allmodifications and equivalents as defined by the appended claims.

DESCRIPTION

The present disclosure relates to the delivery of therapeutic anddiagnostic agents, and more particularly, in certain embodiments, to ananocluster drug delivery platform.

Current drug delivery treatment options can often be ineffective due toinefficient delivery of an active ingredient to a targeted site. Many ofthe current drug delivery systems are limited in their ability orefficiency to access a specifically targeted site. Althoughnanoparticles offer several advantages for delivering drugs (e.g.improved dissolution of low solubility API, intracellular andtranscellular transport, etc.), the use of nanoparticles, for example,can be hindered by the inability to deliver nanoparticles to the site ofdrug action (e.g. dried nanoparticles are too small to depositefficiently in the lungs, can avoid detection by APCs, etc.). Inaddition, nanoparticles are often difficult to handle at an industrialscale and a controlled clustering process may ease handling and allowfacile reconstitution and formulation of nanoparticles or nanoclustersfor delivering drugs.

The nanoclusters of the present invention can be used to deliver activeingredients to a targeted site. The size and distribution of thedisclosed nanoclusters can be designed for a desired route ofadministration and/or for the treatment of a particular disease orcondition. In one aspect, for example, the nanoclusters provide aneffective and efficient drug delivery system that can carry activeingredients to a targeted site via the nanocluster. In certain aspects,a nanocluster is maintained at the targeted site. In other aspects, ananocluster can disperse the active ingredient at the targeted site.Additionally, the nanoclusters can be formulated with the appropriateproperties to carry and controllably release active ingredients to atargeted site.

Nanoclusters

In general, the nanoclusters of the present disclosure comprise aplurality of nanoparticles having a core of nanoparticles arranged suchthat the surface of the nanoparticles contact one another to form athree dimensional structure, the nanoparticles may comprise atherapeutic or diagnostic agent. The nanoclusters of the presentdisclosure, among other things, provide the advantage of particleclusters appropriately sized for delivery of an active ingredient (e.g.,lung, nasal passage, M-cells in the digestive tract, uptake by antigenpresenting cells, etc.) with the benefits of improvements in, forexample, drug solubility, aerosolization, bioavailability, transportthrough biological barriers, and intracellular delivery.

In certain embodiments, a nanocluster of the present disclosure mayinclude an excipient. Excipients and the use of excipients are wellknown in the art. Excipients may be used as, among other things,dispersing agents, agglomerating agents, filling agents, bufferingagents, and the like. Examples of suitable excipients include, but arenot limited to, salts, amino acids, phospholipids, sugar alcohols,surfactants, and the like.

In certain embodiments, a nanocluster of the present disclosure mayinclude nanoparticles from about 1% to about 99% by weight or volume.The nanocluster also may be completely made up of nanoparticles (i.e.,100%).

In certain embodiments, a nanocluster may have a mass median aerodynamicdiameter of from about 0.25 μm to about 20 μm. In other embodiments ananocluster may have a mass median aerodynamic diameter of from about0.5 μm to about 10 μm. In other embodiments a nanocluster may have amass median aerodynamic diameter of from about 3 μm to about 20 μm. Incertain embodiments a nanocluster may have a mass median aerodynamicdiameter of from about 0.5 μm to about 3 μm.

In certain embodiments, a nanocluster of the present disclosure has amass median geometric diameter that is greater than the mass medianaerodynamic diameter, as these particles have low bulk density.

In general, nanoclusters of the present disclosure are non-spherical andoffer a non-continuous surface with irregular shape.

Nanoparticles

Nanoparticles are particles having features on a nanometer scale such asfacets, cubes, angles, and the like. Conceptually, nanoclusters may bedescribed as comprising nanoparticles; however, that is not to implythat the nanoclusters may only be formed by separate nanoparticles.Nanoclusters may be formed without first forming independently stablenanoparticles. In certain embodiments, a nanoparticle is a particlewhose effective diameter measures less than 1,000 nm.

In general, the nanoparticles comprise a therapeutic or diagnostic agent(i.e., an active ingredient).

In general, the active ingredient is associated with the nanoparticle.For example, the active ingredient may be entangled, embedded,incorporated, encapsulated, bound to the surface, or otherwiseassociated with the nanoparticle. In other examples, the nanoparticlecomprises an active ingredient such as a pure drug (e.g., drugsprocessed by crystallization or supercritical fluids, an encapsulateddrug (e.g., polymers), a surface associated drug (e.g., drugs that areabsorbed or bound to the nanoparticle surface), complexed drugs (e.g.,drugs that are associated with the material used to form thenanoparticle). In certain embodiments, the nanoparticle consistsessentially of active ingredient. The nanoparticles may have any type ofstructure.

In certain embodiments, a plurality of nanoparticles is capable ofagglomerating together such that the surfaces of the nanoparticleseffectively contact one another. Such agglomeration may occur throughnoncovalent interactions, such as van der Waals forces, hydrophobicinteractions, Coulombic forces, and the like.

In addition to an active ingredient, the nanoparticles may include othermaterials, such as any organic or inorganic materials suitable forbiological applications. Examples of these materials include, but arenot limited to, surfactants, phospholipids, sugar alcohols,poly(orthoesters), poly(anhydrides), poly(phosphoesters),poly(phosphazenes), poly(lactic-co-glycolic acid) (PLGA), polyesters(such as poly(lactic acid), poly(L-lysine), poly(glycolic acid) andpoly(lactic-co-glycolic acid)), poly(lactic acid-co-lysine), poly(lacticacid-graft-lysine), polyanhydrides (such as poly(fatty acid dimer),poly(fumaric acid), poly(sebacic acid), poly(carboxyphenoxy propane),poly(carboxyphenoxy hexane), copolymers of these monomers and the like),poly(anhydride-co-imides), poly(amides), poly(ortho esters),poly(iminocarbonates), poly(urethanes), poly(organophasphazenes),poly(phosphates), poly(ethylene vinyl acetate) and other acylsubstituted cellulose acetates and derivatives thereof,poly(caprolactone), poly(carbonates), poly(amino acids),poly(acrylates), polyacetals, poly(cyanoacrylates), poly(styrenes),poly(vinyl chloride), poly(vinyl fluoride), poly(vinyl imidazole),chlorosulfonated polyolefins, polyethylene oxide, copolymers,polystyrene, and blends or co-polymers thereof. In certain embodiments,the nanoparticles include hydroxypropyl cellulose (HPC),N-isopropylacrylamide (NIPA), polyethylene glycol, polyvinyl alcohol(PVA), polyethylenimine, chitosan, chitin, dextran sulfate, heparin,chondroitin sulfate, gelatin, and the like, and their derivatives,co-polymers, and mixtures thereof. In certain embodiments thenanoparticles may include any inactive ingredient that is generallyregarded as safe.

Active ingredients may include, but are not limited to, any component,compound, or small molecule that can be used to bring about a desiredeffect. Non-limiting examples of desired effects of the presentinvention include diagnostic and therapeutic effects. For example, adesired effect can include the diagnosis, cure, mitigation, treatment,or prevention of a disease or condition. An active ingredient may alsoaffect the structure or function of body part or organ in a subject.

Active ingredients are known to those skilled in the art. Examples ofactive ingredients that may be used with the nanoclusters of the presentdisclosure include, but are not limited to, medical pharmaceuticals andspecialties such as preventive agents, for example vaccines, diagnosticagents, for example tracers of various types and imaging enhancers,therapeutic agents, for example small molecules (e.g., nucleic acids,proteins, peptides, polypeptides, etc.), drugs, peptides, and radiation,immuno-modulators, vaccine and virus vectors, and combinations of theseclasses. Other suitable active ingredients include respirablenon-medical specialties such as physiochemical agents, for example gasantidotes, biophysical modulators, for example paramagnetics,radioactive compounds for imaging, emitters, for example electromagneticwave emitters, and imaging enhancers.

In certain embodiments, a nanocluster may include more than one activeingredient. For example, a nanocluster may include any combination oftherapeutic agents or diagnostic agents, alone or in combination. Forexample, a nanocluster may include both a therapeutic agent and adiagnostic agent.

Examples of suitable active ingredients which may be included in thenanoclusters of the present disclosure include, but are not limited to,nucleic acids, proteins and peptides, hormones and steroids,chemotherapeutics, antineoplastic agents, NSAIDs, vaccine components,analgesics, antibiotics, CNS agents (e.g., anti-depressants, atypicalantipsychotics, and benzodiazephines), calcium ion channel blockers(e.g., nifedipine), H1 antagonists (e.g., loratadine), and the like.

Examples of suitable nucleic acids that may be used include, but are notlimited to, DNA, cDNA, RNA, iRNA, siRNA, anti-sense nucleic acid,peptide-nucleic acids, oligonucleotides, or nucleic acids that aremodified to improve stability (e.g., phosphorothioates,aminophosphonates or methylphosphonates).

Examples of proteins and peptides that may be used include, but are notlimited to, human growth hormone, bovine growth hormone, vascularendothelial growth factor, fibroblast growth factors, bone morphogenicprotein, tumor necrosis factors, erythropoietin, thrombopoietin, tissueplasminogen activator and derivatives, insulin, monoclonal antibodies(e.g., anti-human epidermal growth factor receptor2 (Herceptin®(trastuzumab), anti-CD20 (rituximab), anti-CD 18, anti-vascularendothelial growth factor, anti-IgE, anti-CD 11a), and theirderivatives, single-chain antibody fragments, human deoxyribonuclease I(domase alfa, Pulmozyme®), type-1 interferon, granulocytecolony-stimulating factor, leuteinizing hormone releasing hormoneinhibitor peptides, leuprolide acetate, endostatin, angiostatin, porcinefactor VIII clotting factor, interferon alfacon-1, pancrelipase(pancreatic enzymes), ovalbumin, and the like.

Examples of hormones and steroids (e.g., corticosteroids) that may beused include, but are not limited to, norethindrone acetate, ethinylestradiol, progesterone, estrogen, testosterone, prednisone and thelike.

Examples of antineoplastic agents that may be used include, but are notlimited to, taxol (paclitaxel), vinblastine, cisplatin, carboplatin,tamoxifen, retanoids, enzyme inhibitors (e.g., COX, VEGF), and the like.

Examples of non-steroidal anti-inflammatory drugs (NSAIDs) that may beused include, but are not limited to, piroxicam, aspirin, salsalate(Amigesic®), diflunisal (Dolobid®), ibuprofen (Motrin®), ketoprofen(Orudis®), nabumetone (Relafen®), piroxicam (Feldene®), naproxen(Aleve®, Naprosyn®), diclofenac (Voltaren®), indomethacin (Indocin®),sulindac (Clinoril®), tolmetin (Tolectin®), etodolac (Lodine®),ketorolac (Toradol®), oxaprozin (Daypro®), and celecoxib (Celebrex®),and the like.

Examples of vaccine components that may be used include, but are notlimited to, Hepatitis B, polio, measles, mumps, rubella, HIV, hepatitisA (e.g., Havrix), tuberculosis, and the like.

Examples of analgesics that may be used include, but are not limited to,aspirin, acetaminophen, ibuprofen, naproxen sodium, and the like.

Examples of antibiotics that may be used include, but are not limitedto, amoxicillin, penicillin, sulfa drugs, erythromycin, streptomycin,tetracycline, clarithromycin, tobramycin, ciprofloxacin, terconazole,azithromycin, and the like.

Examples of anti-depressants that may be used include, but are notlimited to, sertraline (Zoloft®), fluoxetine (Prozac®), paroxetine(Paxil®), citalopram, venlafaxine, fluvoxamine maleate, imipraminehydrochloride, lithium, nefazodone and the like.

Other examples of active ingredient that can be used that may be usedinclude, but are not limited to, sildenafil, acyclovir, gancyclovir,fexofenidine, celecoxib, rofecoxib, androstenedione, chloroquine,diphenhydramine HCl, buspirone, doxazocin mesylate, loratadine,clomiphine, zinc gluconate, zinc acetate, hydrocortisone, warfarin,indinavir sulfate, lidocaine, novacaine, estradiol, norethindroneacetate, medroxyprogesterone, dexfenfluramine, dextroamphetamine,doxycycline, thalidomide, fluticasone, fludarabine phosphate,etanercept, metformin hydrochloride, hyaluronate, tetrazocinhydrochloride, loperamide, ibogaine, clonazepam, ketamine, lamivudine(3TC), isotretinoin, nicotine, mefloquine, levofloxacin, atorvastatin(LipitorZoloft), miconazole nitrate (MonistatZoloft), ritonavir,famotidine, simvastatin, sibutramine HCl monohydride, ofloxacin,lansoprozole, raloxifene, zanamivir, oseltamivir phosphate,4-phenylbutyric acid sodium salt, chlorpromazine, nevirapine,zidovudine, and cetirizine hydrochloride.

Non-limiting examples of additional active ingredients can be found inPhysician's Desk Reference 64th Edition, all of which are incorporatedby reference.

Nanocluster Formulation Variables and Tunability

As mentioned above, the nanoclusters of the present disclosure may havevarious sizes. The size of a particular nanocluster may depend on, amongother things, the nanoparticle type and/or size, excipients, andprocessing conditions. For example, varying processing conditions can beused to create nanoclusters with a broad or narrow size range.Controlling the droplet size in an emulsion, suspension, or solutionsprayed from a nozzle can facilitate the formation of uniformnanoclusters. Varying the solvent and extraction phase, temperature,humidity, and other conditions, as well as the properties of thenanoparticles, can control the morphology of the nanocluster. Shear alsomay be used to control the morphology (e.g., size and/or shape) ofnanoclusters. Varying processing times (e.g., hold times) also may leadto, for example, larger or smaller nanoclusters.

The tunability of the size of the nanocluster may be beneficial inseveral applications. For example, broadening the size distribution mayimprove the flow properties of a powder, thus facilitatingmanufacturing. Having a narrow distribution with a mass medianaerodynamic size range of about 0.5 to about 3 micrometers may be usefulin the delivery of drugs via the inhalation route to the distal lung.Changing the median aerodynamic size may favor deposition in aparticular region of the pulmonary system. Optimizing the size anddistribution of the nanocluster to achieve optimum dissolution,compactability, and strength of tablet formulations may be useful in thedevelopment of tablets for oral route of administration.

Methods for Preparing Nanoclusters

The present disclosure provides, in certain embodiments; methods forpreparing a nanoclusters. In general, such methods comprise forming ananocluster comprised of a core of nanoparticles arranged such that thesurface of the nanoparticles contact one another, wherein thenanocluster has a mass median aerodynamic diameter of from about 0.25 μmto about 20 μm.

In certain embodiments, the nanoclusters may be formed usingpost-processing agglomeration followed by recovery of nanoclusters. Inother embodiments, the nanoclusters may be formed using in-processingagglomeration followed by recovery of nanoclusters. Post-processingagglomeration generally comprises providing a colloidal dispersion ofnanoparticles, agglomerating the nanoparticles, and recovering thenanoclusters. In-processing agglomeration generally comprises processingan active ingredient under conditions such that an agglomerate forms,where such agglomerate is already a nanocluster or from which ananocluster may be recovered.

Post-Processing Agglomeration

As mentioned above, post-processing agglomeration generally comprisesproviding a colloidal dispersion of nanoparticles and agglomerating thenanoparticles.

In certain embodiments, the colloid dispersion of nanoparticles may beformed using attrition (e.g., size reduction of bulk solids) and/orprecipitation (e.g., the formation of a suspension of nanoparticles froma solution).

Attrition may be accomplished using techniques such as milling (e.g.,impact milling, jet milling, ball milling, wet milling, and the like),hydrodynamic cavitation, high shear fluid processing, homogenization,sonication, and the like. For example, in certain embodiments,unprocessed active ingredient may be processed to reduce particle size.In general, such embodiments comprise suspending unprocessed activeingredient in a suitable non-solvent to form a suspension and subjectingthe unprocessed active ingredient to attrition to decrease the activeingredient's particle size. In some embodiments, the active ingredient'sparticle size may be reduced to an effective diameter less than 1,000nm.

The non-solvent may be any liquid in which the active ingredient issubstantially insoluble and/or which is capable of forming a suspensionof active ingredient particles. Examples of suitable non-solventsinclude, but are not limited to water, acetone, methylene chloride,ethanol, hexane, and the like. The particular non-solvent chosen willdepend upon, among other things, the solubility of the active ingredientand effect of solvent on surface energy.

The non-solvent also may include a material capable of changing thesurface energy of the particles, for example, to allow the particles toagglomerate. Examples of such materials include agglomerating agents(e.g., surfactants, co-solvents, and salts). Such materials may bepresent in an amount sufficient to allow the particles to agglomerateand the amount may be tailored to achieve a desired size of theagglomerate. Examples of suitable surfactants include, but are notlimited to, cetyl alcohol, PL, PVA, PVP K90, lecithin, leucine, Span 85,Pluronics, and other surfactants that are generally regarded as safe,and the like. Examples of suitable co-solvents include, but are notlimited to, water, methanol, hexane, ethanol, acetone, acetonitrile,octanol, and the like. Examples of suitable salts include, but are notlimited to, sodium chloride, calcium chloride, magnesium sulfate, andthe like. The particular agglomerating agent chosen will depend upon,among other things, the solubility of the active ingredient and theaffect of the agglomerating agent on the surface energy of theparticles.

As mentioned above, precipitation may be used to form a colloiddispersion of nanoparticles. Precipitation may be accomplished usingtechniques such as solvent/anti-solvent, cryo-based precipitation, flashprecipitation, sonoprecipitation, and the like. For example, in certainembodiments, an active ingredient may be dissolved to form a solution ofthe active ingredient and precipitated out of solution in a non-solventto form a colloid suspension of active ingredient.

Once a colloid of active ingredient is formed it is at least partiallyagglomerated into nanoclusters. By way of explanation, agglomeration mayoccur as a result of a change in the surface energy of the particlesthat allows the particles in the colloid to agglomerate. In someembodiments, agglomeration may occur as a result of a change in particlesize during processing. By way of explanation, smaller sized particlestypically demonstrate an increase in surface energy and mobility, andthese properties can drive assembly of particles into nanoclusters. Inother embodiments, entropic effects such as water exclusion may be usedto agglomerate a colloid of the active ingredient. In still otherembodiments, desolvation of the colloid may be used to formagglomerates. In some embodiments, agglomerates may be formed throughthe action of a material capable of changing the surface energy ofparticles that may be present in the non-solvent (e.g., agglomeratingagent). For example, an agglomerating agent may be added to a colloid.

In-Processing Agglomeration

As mentioned above, in-processing agglomeration generally comprisesprocessing an active ingredient under conditions such that anagglomerate forms. In certain embodiments, the processing uses attritionand/or precipitation, as described above. In-processing differs frompost-processing in that agglomerates form without first forming a stablecolloid. In some embodiments, the change in surface energy of thecolloid occurs during processing. In certain non-limiting examples,in-processing agglomeration may be the result of changes in surfaceenergy of particles and/or a change in particle size.

Recovery of NanoClusters

In certain embodiments, it may be beneficial to recover the nanoclusterfrom suspension.

In certain embodiments, in which a non-solvent is used, a nanoclustermay be recovered by removing the non-solvent. Examples of techniquessuitable for removing the non-solvent and recovering nanoclustersinclude, but are not limited to, evaporation, vacuum drying,spray-drying, freeze drying, spray freeze-drying, and lypholization.

In some embodiments, nanoclusters of the present disclosure may beformed through the action of shear. For example, nanoclusters may beformed by allowing a plurality of nanoparticles to agglomerate togetherand applying shear to the agglomerated nanoparticles to form ananocluster. Examples of techniques suitable for applying shear includehomogenization, sonication, wet milling, high shear flow processing, andthe like. In certain embodiments, shear may be applied upon exiting adevice for administration to a subject (e.g., an inhaler). For example,nanoclusters may be formed from agglomerated nanoparticles as they exita metered dose inhaler and/or a dry powder inhaler.

The size of the resulting nanocluster also may be controlled bytailoring the recovery conditions. For example, more or less shear maybe used to create nanoclusters in a desired size range. Similarly, amaterial capable of changing the surface energy of the particles (e.g.,hydrophobicity) may be used to form nanoclusters in a desired sizerange.

The present disclosure provides, in certain embodiments, methods forpreparing nanoclusters comprising: forming a nanocluster comprised of acore of nanoparticles arranged such that the surface of thenanoparticles contact one another, wherein the nanocluster has a massmedian aerodynamic diameter of from about 0.25 μm to about 20 μm.

The present disclosure provides, in certain embodiments, methods forpreparing nanoclusters comprising: providing a colloidal dispersion ofnanoparticles, agglomerating at least a portion of the nanoparticles toform nanoclusters, and optionally recovering the nanoclusters.

The present disclosure provides, in certain embodiments, methods forpreparing nanoclusters comprising: suspending unprocessed activeingredient in a non-solvent to form a suspension; attrition of theactive ingredient to form a colloidal dispersion of nanoparticles;agglomerating at least a portion of the nanoparticles to formnanoclusters; and optionally recovering nanoclusters.

The present disclosure provides, in certain embodiments, methods forpreparing nanoclusters comprising: suspending unprocessed activeingredient in a non-solvent to form a suspension; attrition of theactive ingredient to form a colloidal dispersion of nanoparticles; andallowing nanoclusters to form in the suspension.

The present disclosure provides, in certain embodiments, methods forpreparing nanoclusters comprising: precipitating active ingredient in asuitable non-solvent; obtaining a colloidal suspension of precipitatedactive ingredient particles; agglomerating at least a portion of thecolloidal suspension of precipitated active ingredient particles to formnanoclusters; and recovering the nanoclusters.

The present disclosure provides, in certain embodiments, methods forpreparing nanoclusters comprising: precipitating active ingredient in asuitable non-solvent; obtaining a colloidal suspension of precipitatedactive ingredient particles; and allowing nanoclusters to form in thesuspension.

In certain embodiments, methods for preparing a nanocluster comprisinginclude: precipitating active ingredient particles in a suitablenon-solvent; and allowing at least a portion of the precipitated activeingredient particles to agglomerate into nanoclusters.

In certain embodiments, it is contemplated that the nanoclusters may beprepared in a solution without using spray and/or freeze dry techniques.It is also contemplated that the nanoclusters may be recovered from thesolution by using freeze dry or spray dry techniques that are known tothose of skill in the art. As noted throughout this disclosure, thenanocluster can be included within a composition. The composition can beformulated into a suspension, a liquid, a spray, an aerosol, a drypowder, or solid dosage form, among other things.

The present disclosure provides, in certain embodiments, methods forpreparing a nanocluster comprising: (i) obtaining a plurality ofnanoparticles; (ii) obtaining a dispersion material (when desired); and(iii) admixing the products of steps (i) and (ii), wherein the admixtureis formulated into a nanocluster. In certain embodiments, obtaining aplurality of nanoparticles comprises: (i) obtaining an aqueoussuspension of nanoparticles; (ii) emulsifying the suspension into anon-aqueous phase; (iii) allowing water in the aqueous suspension toabsorb into the non-aqueous phase; (iv) allowing the nanoparticles toagglomerate together; and (v) retrieving the agglomerated nanoparticles.In other embodiments, obtaining a plurality of nanoparticles includes:(i) obtaining a non-aqueous suspension of nanoparticles; (ii)emulsifying the suspension into an aqueous phase; (iii) allowing liquidin the non-aqueous suspension to absorb into the aqueous phase; (iv)allowing the nanoparticles to agglomerate together; and (v) retrievingthe agglomerated nanoparticles. For example, a colloidal suspension ofnanoparticles may also be obtained in deionized water which issubsequently emulsified into octanol. Water in the dispersed dropletsmay then absorb into the octanol phase. Nanoparticles can pack togetheras water is extracted from individual droplets until an agglomerate ofnanoparticles remains. The size of the droplet, in certain embodiments,may serve as a template for controlling the size of the resultingnanoclusters depending on the concentration of nanoparticles within thedroplet.

As noted throughout this disclosure, the nanocluster can be includedwithin a composition. The composition can be formulated into a liquid, aspray, an aerosol, a suspension, and a dry powder, among other things.For example, the composition may be formulated for use in a dry powderinhaler (DPI) or a pressurized metered dose inhaler (pMDI).

In certain embodiments, nanoclusters of the present disclosure may beformed into a dry powder formulation. For example, nano-suspensions ofan active ingredient may be prepared using precipitation techniquesknown in the art from a solution of the drug. Agglomerates may then beprepared by adding an agglomerating agent and allowing the suspension toincubate for a period of time. Following the agglomeration, the solventmay be evaporated. The dry nanoclusters are then placed into a freezerdryer to remove the last of the solvent. This process allows thelyophilized powder to be conveniently stored for subsequent use.

Once the dry powder has been formulated, the drug may be administered inits existing form with a dry powder inhaler (DPI). However, one featureof the present disclosure is that the same dry powder may be resuspendedand administered as a nebulized solution. The resuspension of the drypowder in the present disclosure simply requires the addition of water.In certain embodiments, the dry powder may be resuspended in a ratio ofabout 50 mg of lyophilized powder to about 10 mL of water. In otherembodiments, the dry powder may be resuspended in any other ratio suchthat it is suitable for nebulization.

Once resuspended, the solution of dry powder in water may be nebulizedaccording to techniques known in the art. Suitable nebulizers include,but are not limited to, jet nebulizers, vibrating mesh, and sonicating.In certain embodiments, the nebulized solution comprises droplets in thedesired respirable range usually but not limited to between 0.5-4.0 μm.

Pharmaceutical Compositions and Routes of Administration

In another embodiment, the present disclosure provides a compositioncomprising a nanocluster. In certain embodiments, the composition (e.g.,pharmaceutical composition) may comprise a plurality of nanoclusters;and, the nanoclusters may be identical to similar nanoclusters or thenanoclusters may be nanoclusters with different characteristics (e.g.,different active ingredients attached, different shapes, differentsizes, and the like). The compositions comprising nanoclusters may beformulated with a pharmaceutically acceptable carrier.

Certain embodiments of the present disclosure include methods oftreating, preventing, or diagnosing a particular disease or condition byadministering the disclosed nanoclusters to a subject. In manyinstances, the nanoclusters are administered alone or can be includedwithin a pharmaceutical composition. An effective amount of apharmaceutical composition, generally, is defined as that amountsufficient to ameliorate, reduce, minimize or limit the extent of thedisease or condition. More rigorous definitions may apply, includingelimination, eradication, or cure of the disease or condition.

Pharmaceutical Compositions

Pharmaceutical compositions of the present disclosure can include ananocluster of the present disclosure. The phrases “pharmaceutical orpharmacologically acceptable” may include, but is not limited to,molecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to a subject, suchas, for example, a human. The preparation of a pharmaceuticalcomposition is generally known to those of skill in the art. Moreover,for animal (e.g., human) administration, it is preferred that thepreparations meet sterility, pyrogenicity, general safety and puritystandards as required by FDA Office of Biological Standards.

“Therapeutically effective amounts” are those amounts effective toproduce beneficial results in a subject. Such amounts may be initiallydetermined by reviewing the published literature, by conducting in vitrotests, and/or by conducting metabolic studies in healthy experimentalanimals. Before use in a clinical setting, it may be beneficial toconduct confirmatory studies in an animal model, preferably a widelyaccepted animal model of the particular disease to be treated.

The actual dosage amount of a composition of the present disclosureadministered to a subject can be determined by physical and/orphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient, and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

The compositions of the present invention may include different types ofpharmaceutically acceptable carriers depending on whether it is to beadministered in solid, liquid, or aerosol form, and whether it needs tobe sterile for such routes of administration as injection.“Pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, surfactants, antioxidants, preservatives(e.g., antibacterial agents, antifungal agents), isotonic agents,absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes),propellants, and combinations thereof. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithinand/or leucine; by the maintenance of the required particle size bydispersion in carriers such as, for example liquid polyol or lipids; bythe use of surfactants such as, for example hydroxypropylcellulose; orcombinations thereof such methods. In many cases, it will be preferableto include isotonic agents, such as, for example, sugars, sodiumchloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols, or inhalants in the present invention. Such compositions aregenerally designed to be compatible with the target tissue type. Forexample, nasal solutions are usually aqueous solutions designed to beadministered to the nasal passages in drops or sprays. Nasal solutionsare prepared so that they are similar in many respects to nasalsecretions, so that normal ciliary action is maintained. Thus, incertain embodiments, the aqueous nasal solutions usually are isotonic orslightly buffered to maintain a pH of about 5.5 to about 6.5. Inaddition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments, the compositions are prepared for administrationby such routes as oral ingestion. In these embodiments, the solidcomposition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet.Preferred carriers for oral administration comprise inert diluents,assimilable edible carriers or combinations thereof. In other aspects ofthe invention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, and may comprise, for example, at least oneactive agent, a sweetening agent, a preservative, a flavoring agent, adye, a preservative, or combinations thereof.

In certain embodiments, an oral composition may comprise one or morebinders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample, peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with severalof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

Routes of Administration

The pharmaceutical compositions of the present disclosure can beadministered intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intranasally,intravitreally, intravaginally, intrauterinely, intrarectally,intrathecally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, locally, inhalation (e.g., aerosol inhalation), nebulization,injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in creams, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art.

Combination Therapies

In order to increase the effectiveness of a treatment with thenanoclusters of the present invention, it may be desirable to combinethese nanoclusters with other therapies effective in the treatment of aparticular disease or condition. The pharmaceutical compositions of thepresent disclosure, for example, can precede or follow the other agenttreatment by intervals ranging from minutes to weeks.

Nanoclusters for Pulmonary Administration

Pharmaceutical compounds are delivered to the lungs for a variety ofreasons. Certain compounds are administered to the lungs for theirtherapeutic effect; other compounds are administered for diagnosticpurposes. Delivery to the peripheral lung is important for effective useof many therapeutic and diagnostic agents. A major deterrent toutilizing the lung for systemic drug delivery is the inability toefficiently deliver drugs to the lung periphery (alveolar region) wherefree exchange with the circulatory system occurs. Current techniques(e.g. milling or spray drying) often produce broad particle sizedistributions, which translates into poor pulmonary depositionefficiency. Over the past 30 years, researchers have found that about 2μm particles deposit with high efficiency to the deep lung avoidingdeposition in the oropharangyal cavity common for larger particles (>5μm) and exhalation common for submicron particles. Unfortunately,particles around this size and smaller suffer from a propensity toagglomerate; therefore, researchers have developed low density particlesas a method to effectively deliver drugs to the deep lung. Theseparticles possess large geometric diameters, but due to their lowdensity, exhibit much smaller aerodynamic diameters as described by theequation, d_(aero)=d_(p)[(ρ/ρ_(ref))/γ]0.5, where d_(aero) is theaerodynamic particle diameter, d_(p) is the geometric particle diameter,ρ is the particle density, ρ_(ref) is a reference density (typically 1g/cm³) and γ is a shape factor (typically 1 for a sphere). Large, lowdensity particles possessing geometric diameters greater than about 15to 20 μm are able to avoid uptake and clearance by macrophages; however,these cells may still be recruited to the site of particle deposition.

Nanoclusters of the present disclosure may be introduced into the lungin one of several different ways. In certain embodiments, nanoclustersmay be formulated into a dry powder that is inhaled into the lung. Inother embodiments, nanoclusters may be dissolved into a solution that issubsequently nebulized, or converted into a fine mist, prior toinhalation. Such delivery modes for nanoclusters and nanoclusterformulations may be useful for treating for example, cystic fibrosis,asthma, and lung cancer, as well as for delivering large immunoglobulinsor genetic material systemically by crossing the lung epithelium.

Conventional nebulized suspensions consist of large insolubleparticles >2 microns in aerodynamic size that cannot be carried by manyof the smaller, more respirable droplets generated by a jet nebulizer.The nanocluster formulations of the present disclosure improve the massof drug carried in the fine particle fraction of aerosolized droplets,improving delivery efficiency and carrying a greater mass of drug out ofthe nebulizer and into the lung. In this way, smaller drug doses may becapable of delivering a greater percentage of active ingredient in therespirable fraction of droplets generated using nebulization. Improvingthe fine particle fraction may provide significant therapeutic benefit,among other things, by distributing drug more uniformly throughout thecentral and peripheral lung areas.

Accordingly, in certain embodiments, the present disclosure relates tothe pulmonary delivery of nanoclusters as either a dry powder or anebulized solution. Embodiments of the present disclosure includeparticular nanoclusters of active ingredients that function essentiallythe same way in either formulation. This outcome can be achieved bypreparing nanoparticles according the techniques described above,causing the nanoparticles to agglomerate into nano-clusters, dewateringthe agglomerated nano-clusters into a dry powder, and optionallysuspending the dry powder in a nebulization liquid. This dry powder maybe conveniently stored and subsequently used in either its powder formor after being resuspended and nebulized. For example, such formulationsmay be used in dry powder inhalers, nebulizers, or in-line ventilators.

The pulmonary administration of a dry powder formulation is oftenlimited by the physiological response of the respiratory tract, i.e.,the cough reflex. Traditionally, 20 mg of dry powder is the largestamount of dry powder that is usually administered, although someresearch claims that 50 mg may be reached. By administering thenanoclusters of the present disclosure as a nebulized solution, 100 mgmay be achieved. Additionally, nebulized solutions frequently face lessregulatory hurdles than their dry powder analogs. By producing aformulation with the same characteristics as a dry powder or a nebulizedsolution, the present disclosure facilitates the development andpulmonary administration of the drugs.

The present disclosure, according to certain embodiments, also providesa single formulation that is optimized as a nebulized and dry powderformulation simultaneously. Such embodiments may allow the developmentof aerosol formulations where the preclinical and early stage clinicalwork done by nebulization is able to be used when the formulation istranslated into a dry powder formulation.

In certain embodiments, a dry powder nanocluster formulation producedaccording to the methods of the present disclosure may be suitable foruse in a dry powder inhaler, but may be reconstituted as a nebulizedformation simply though the addition of a suitable solution (e.g.,saline, deionized water, and the like). In certain embodiments, theaddition of a dry powder nanocluster formulation does not substantiallydecrease the vapor pressure of the solution. The components of theformulation are largely insoluble and would not be substantiallydissolved by the aqueous nebulization liquid as to not impact thecolligative properties of the aqueous system. Similarly, the evaporationrate of the nebulized nanocluster formulation is similar to the solutionwithout a drug. In some embodiments, the addition of the dry powdernanocluster formulation does not materially increase the viscosity ofthe solution. As with, for example, beta cyclodextrin enabled nebulizedformulations, the beta cyclodextrins substantially dissolve in theaqueous liquid significantly increasing the viscosity of the solution.

In certain embodiments, nanoclusters of the present disclosure may beadministered to a subject through medical tubing (e.g., ventilatortubing, endotracheal tubes, and the like).

In certain embodiments, the nanoclusters that are delivered to the lungsin the present disclosure may be consumed by macrophages. As these cellsreturn to the lymph nodes, they carry the nano-cluster and facilitate apulmonary delivery of drugs to the lymph nodes.

EXAMPLES

The following examples are included to demonstrate certain non-limitingaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors to function well in thepractice of the invention. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Nanoclusters with Responsive Dispersion

Nanocluster Formation: Nanoclusters of the present disclosure can beprepared by the following procedure: Two syringe pumps (HarvardApparatus 4400 and Isco) are connected to the inner and outer ports of acoaxial nozzle to pass a colloidal suspension of nanoparticles (e.g.colloid suspension in aqueous solution and 1-octanol (Fisher Scientific)as droplet carrying liquid, respectively). The two immiscible liquidsare injected at appropriate flows to produce monodisperse aqueousdroplets, which contain the colloidal suspension of nanoparticles, inthe octanol phase. The nanoclusters are formed after water in thedroplets dissolves into 1-octanol resulting in packing of thenanoparticles into a spherical structure (FIG. 1). Nanoclusters are thenwashed with ethanol to remove residual 1-octanol and can be freeze driedfor analysis. Similar results may be achieved by simply adding thenanoparticle suspension to the octanol phase and stirring to form aprimary emulsion.

In one embodiment, the inventors coated silica nanoparticles coated withpoly(N-vinylformamide) and cross-linked this polymer with a hydrolyzablecross-linker (2-bis[2,2′-di(N-vinylformamido)ethoxy]propane) to formnanoclusters that dispersed in response to a decrease in pH (FIG. 2).This set-up demonstrates the ability to disperse nanoclusters inresponse to environmental cues.

The clustered nanoparticles were slightly different in appearance due tothe presence of the polymer, but the size distribution remainedconsistent with previous experiments. The nanoclusters were dispersedinto aqueous solution as a function of time and pH (FIG. 3). A turbidityassay was used to measure optical density at 480 nm over time, theopacity of the solution indicating the relative dispersion of theclusters into constituent nanoparticles. The dispersion of thenanoclusters could also be visually tracked over time (FIG. 3B). Sizeanalysis of the solution phase of dispersed nanoclusters via laser lightscattering indicated that polydisperse agglomerates of nanoparticleswere liberated. These agglomerates further dispersed into individualnanoparticles over time (FIG. 3C).

Example 2 Self-Assembled Nanoclusters

Nanoparticle Formation: PLGA nanoparticles were prepared by a modifiedemulsion/solvent extraction method using different polyelectrolytecoating materials to control surface charge (Table 1). Polyvinylamine(PV Am) was used as a cationic coating material and was synthesizedaccording to methods known in the art. Polyethylene-alt-maleic acid(PEMA) was synthesized by hydrolysis of the anhydride from of thispolymer as adapted from methods reported previously. The resultingpolyelectrolyte-coated PLGA nanoparticles possessed excellent uniformityand high surface charge (Table 1). Each nanoparticle formulation wasanalyzed for size and zeta potential using dynamic light scattering andconductivity measurements (Brookhaven ZetaPALS), respectively, in theappropriate media (water or organic). Studies confirmed the maintenanceof particle surface charge upon lyophilization and after more than oneweek of incubation at 37° C., pH 7.4 (data not shown). PVAm-coatednanoparticles were notably larger than PEMA-coated nanoparticles forthis experiment; however, this size is readily controlled. Nanoparticlescan be made by using reported techniques, for example; emulsionpolymerization, emulsion solvent extraction, reverse emulsions of thesame, precipitation, crystallization, freeze drying, spray freezedrying, salting out, and the like.

TABLE 1 PLGA nanoparticle properties PLGA Nanoparticle Size (nm) Size(nm) Zeta potential (mV) PVAm-coated 498.5 ± 8.4  +30.7 ± 1.0PEMA-coated 262.7 ± 11.3 −52.3 ± 1.2

Nanocluster Formation: Nanoparticle clusters were produced by slowaddition of 3 mL of PVAm-coated nanoparticles into 10 mL of PEMA-coatednanoparticles under gentle stirring. Nanocluster formation was inducedby electrostatic self-assembly of the oppositely charged nanoparticles.Increasing the concentration of mixed nanoparticles resulted in acorresponding increase in the cluster diameter (FIG. 4A). The geometricsize distribution of nanoclusters was determined in aqueous solution(Isoton) using a Coulter Multisizer III. Geometric size distributionswere relatively broad exhibiting standard deviations that were 60-70% ofthe average geometric diameter. The aerodynamic size distributions weredetermined from freeze dried nanoclusters using time of flightmeasurements obtained by an Aerosizer LD. Nanocluster aerodynamic sizedistributions were narrower than the geometric size distributions asindicated by the increased volume percent (FIG. 4B) and decreasedstandard deviations (35-60% of the mean; Table 2). Free PEMA-coatednanoparticles were detected as a rising tail in the geometric sizedistributions, but were not detected in the aerodynamic sizedistributions. In addition, few free nanoparticles were observed inscanning electron micrographs (FIG. 4C) indicating that nanoparticlesthat were not associated with nanoclusters in solution bound tonanoclusters during lyophilization.

TABLE 2 Dependence of the size of nanocluster on the concentration ofPLGA nanoparticles. PLGA NP conc. (mg/ml) (−)* (+)** (−) (+) (−) (+) (−)(+) 0.68 0.72 1.36 1.44 2.04 2.16 2.72 2.82 d_(geo) (μm) 7.4 ± 5.1 8.7 ±5.6 9.4 ± 5.6 13.7 ± 8.3  d_(aero) (μm) 2.4 ± 1.5 3.0 ± 1.6 3.4 ± 1.64.4 ± 1.6 d_(geo)/d_(aero) 0.33 ± 0.29 0.35 ± 0.29 0.36 ± 0.29 0.32 ±0.19 Calculated ρ 0.11 ± 0.09 0.12 ± 0.08 0.13 ± 0.08 0.10 ± 0.04(g/cm³) Fine Particle 1.00 0.80 0.75 0.50 Fraction *(−): PLGAnanoparticles with negative surface charge (PEMA-coated) **(+): PLGAnanoparticles with positive surface charge (PVAm-coated) ¹Densitycalculated using d_(geo) = d_(p), ρ_(ref) = 1 g/cm³ and γ = 1. ²FineParticle Fraction defined as the fraction of dry particles with d_(aero)(μm) < 5 μm.

Structural analysis of nanoclusters revealed a low density, web-likemorphology. Scanning electron micrographs showed that freeze driednanoparticle clusters possessed a large amount of porosity (FIG. 4C).The density of each nanocluster formulation was calculated from thegeometric and aerodynamic diameters (Table 2). Although nanocluster sizeincreased with increasing nanoparticle concentration, the calculateddensity was essentially the same for each. The calculated densities ofnanoclusters were quite low (˜0.15 g/cm³) and yielded a very high fineparticle fraction (d_(aero)<5 μm) for most formulations. The use of ahydrated geometric diameter, dehydrated aerodynamic diameter, and theassumed shape factor of γ=1 (i.e., a sphere) should be noted wheninterpreting this density calculation.

Laser scanning confocal microscopy was utilized to gain more insightinto the nanocluster structure and formation mechanism. PVAm-coatednanoparticles were labeled with a green fluoroisothiocyanate (FITC) dyeand PEMA-coated nanoparticles were labeled with a red rhodamine dye.Detailed analysis of two nanocluster particles indicated that bothPVAm-coated (FIG. 5A and FIG. 5D) and PEMA-coated (FIG. 5B and FIG. 5E)nanoparticles were present throughout the entire nanocluster structure.

Example 3 Preparation of Nifedipine and Loratadine Nanoparticles

The following includes examples of preparing various non-limitingnifedipine and loratadine nanoparticles.

1020 nm Nifredipine nanoparticle—Nifedipine (50 mg) was dissolved in 3ml of methylene chloride and rapidly mixed into 0.125%Cetyltrimethylammonium bromide (CTAB) solution (30 mL) and sonicated for60 sec. The particle suspension was placed into a hood for two hours toevaporate the methylene chloride. The resulting nanoparticle had aparticle size of 1020 nm and a polydispersity of 0.24.

660 nm Nifedipine nanoparticle—Nifedipine (50 mg) was dissolved in 3 mlof methylene chloride and rapidly mixed into 0.5% CTAB solution (30 mL)and sonicated for 60 sec. The particle suspension was placed into a hoodfor two hours to evaporate the methylene chloride. The resultingnanoparticle had a particle size of 660 nm and a polydispersity of 0.17.

480 nm Nifedipine nanoparticle—Nifedipine (50.2 mg) was dissolved in 3ml of ethanol and rapidly mixed into 0.5% CTAB solution (30 mL) andsonicated for 60 sec. The particle suspension was placed into a hood fortwo hours to evaporate the ethanol. The resulting nanoparticle had aparticle size of 480 nm and a polydispersity of 0.12.

2373 nm Nifedipine nanoparticle—Nifedipine (30 mg) was dissolved in 2 mlof ethanol and rapidly mixed into 0.3% Pluronic F-68 solution (30 mL)and homogenized at 15,000 rpm for 60 sec. The particle suspension wasplaced into a hood for two hours to evaporate the ethanol. The resultingnanoparticle had a particle size of 2373 nm and a polydispersity of0.09.

897 nm Nifedipine nanoparticle—Nifedipine (30 mg) was dissolved in 2 mlof ethanol and rapidly mixed into 0.6% Pluronic F-68 solution (30 mL)and homogenized at 15,000 rpm for 60 sec. The particle suspension wasplaced into a hood for two hours to evaporate the ethanol. The resultingnanoparticle had a particle size of 897 nm and a polydispersity: 0.07.

639 nm Nifedipine nanoparticle—Nifedipine (30 mg) was dissolved in 2 mlof ethanol and rapidly mixed into 0.9% Pluronic F-68 solution (30 mL)and homogenized at 15,000 rpm for 60 sec. The particle suspension wasplaced into a hood for two hours to evaporate the ethanol. Resultingnanoparticle had a particle size of 639 nm and a polydispersity of0.005.

391 nm Loratadine nanoparticle—Loratadine (10 mg) was dissolved in 1 mlof ethanol and rapidly mixed into 0.9% Pluronic F-68 solution (10 mL)and homogenized at 15,000 rpm for 60 sec. The particle suspension wasplaced into a hood for two hours to evaporate the ethanol. The resultingnanoparticle had a particle size of 391 nm and a polydispersity of0.005.

Example 4 Preparation of Nifedipine Nanoparticle Clusters

This example provides a non-limiting embodiment of the present inventionwhere the nanoparticle is pure nifedipine (a calcium channel blockerthat treats high blood pressure). The nanoparticle is coated with acationic surfactant (CTAB). A polyanion (sodium alginate) couples withthe CTAB which induces nanocluster formation.

Preparation of nifedipine nanoparticles: Nifedipine (50 mg) wasdissolved in methylene chloride (3 ml). The solution was pouredcompletely into a CTAB concentration-known aqueous solution (Table 3).The solution was sonicated for 60 sec. Subsequently, the particlesuspension was placed into a hood for two hours to evaporate themethylene chloride. The suspension was diluted to 1 mg/ml. FIG. 6 is ascanning electron microscope (SEM) image of a population of nifedipinenanoparticles.

TABLE 3 Geometric size and aerodynamic diameters of clusters*Aerodynamic Aerodynamic diameter (before diameter (after Conc. of CTABGeometric size grinding) (mass grinding) (mass (wt %)V_(nifedipine)/V_(algenic acid) (μm) median, μm) median, μm) 0.125 2:128.11 ± 8.33  3.313 ± 1.868 3.321 ± 1.763 1:1 22.84 ± 11.64 3.814 ±1.811 4.133 ± 1.829 1:2 29.27 ± 11.47 4.219 ± 1.597 4.234 ± 1.836 1:323.31 ± 13.4  3.397 ± 1.858 3.702 ± 1.844 0.25 2:1 27.24 ± 11.42 3.775 ±1.804 3.467 ± 2.025 1:1 29.49 ± 12.36  3.98 ± 1.868 4.135 ± 1.803 1:223.36 ± 13.48 4.217 ± 1.874 4.312 ± 1.926 1:3 23.82 ± 10.50 3.520 ±1.989 4.006 ± 1.903 0.4 2:1 26.39 ± 12.76 3.819 ± 1.786 4.715 ± 1.3971:1 33.74 ± 13.85 4.156 ± 1.769 3.840 ± 1.942 1:2 30.97 ± 14.31 3.793 ±1.866 3.973 ± 1.876 1:3 23.72 ± 15.70 / / *Concentration: particlesuspension: 1 mg/ml; Algenic acid: 1 mg/mlV_(nifedipine)/V_(algenic acid)

Preparation of nifedipine nanoparticle clusters: Algenic acid aqueoussolution (10 ml, 1 mg/ml) was poured into nifedipine nanoparticleaqueous suspension (10 ml, 1 mg/ml) and the mixture was homogenized witha homogenizer (about 2000 rpm) for 2 min. Dry Nifedipine nanoparticleclusters were obtained by freeze-drying. FIG. 7 is a SEM image ofnifedipine nanoparticle clusters.

Example 5 Nanocluster Comprising Ovalbumin

This example provides a non-limiting embodiment of the present inventionwhere the nanoparticle is a biodegradable polymer (PLGA) coated with acationic lipid (DOTAP). Ovalbumin couples to the surface of the coatednanoparticle which induces nanocluster formation.

Preparation of nanoparticles: PLGA nanoparticles were prepared using amodified emulsion-solvent evaporation technique (Kazzaz et al., 2000;Mainardes et al., 2005, both of which are incorporated by reference). Acationic surface charge was incorporated using the lipid1,2-dioleoyl-3-trimethylammonium-propane (DOTAP; Avanti Polar Lipids,Inc.; Alabaster, Ala.) as the coating material. 3 mL PLGA (0.41 dL/ginherent viscosity; Lactel; Pelham, Ala.) dissolved in anacetone/methanol mixture (5/1) at 1.67% (w/v) was added to 25 mL DOTAP(50 μM) and sonicated at 50% power using a sonic dismembrator (FisherScientific; Pittsburgh, Pa.) for 60 sec on ice. This was repeated for atotal of 6 batches. The batches were combined and stirred at moderatespeed in the hood overnight to evaporate the solvent. The particles werecrudely filtered through a KimWipe and washed three time's with ˜15 mLdistilled, deionized water using an Amicon Ultra-15 centrifugal filterunit (Millipore; Billerica, Mass.; F=863 g). The washed nanoparticleswere sonicated in a water bath for 15 min and again filtered through aKim Wipe to remove any large agglomerates. The resulting particles werethen characterized using a Zeta Potential Analyzer (BrookhavenInstruments; Holtsville, N.Y.) to measure particle size and surfacecharge (ζ): the nanoparticles had an average size of 343.0±8.6 (nm), apolydispersity of 0.232±0.022, and a zeta potential of 36.44±0.56 (mV).The nanoparticle suspension was diluted in 1 mM sodium nitrate solutionfor surface charge measurements.

Spontaneous nanocluster formation of nanoparticles with ovalbumin:Ovalbumin was used as a model protein. Three solutions containingapproximately 0.4, 1.5 and 2.5 mg/mL ovalbumin were prepared inphosphate buffered saline (PBS), and the exact concentration of eachsolution was determined using UV absorbance spectroscopy (Table 4).Using three labeled, 15 mL centrifuge tubes, 6 mL DOTAP nanoparticlesand 1 mL ovalbumin solution were added. The samples were tumbled gentlyon an end-over-end tube rotator for 45 min at 4° C. The resultingnanoclusters were analyzed using a Multisizer 3 Coulter Counter (BeckmanCoulter, Inc.; Fullerton, Calif.) to measure their geometric diameter.The nanoclusters were lyophilized using a Labconco bench-top lyophilizer(Kansas City, Mo.) and further characterized to determine theaerodynamic diameter (Aerosizer; Amherst Process Instruments Inc.) andmorphology (SEM) (Table 5). FIG. 8 illustrates the geometric diameter ofthe DOTAP/PLGA nanoparticles with ovalbumin.

TABLE 4 Concentration of ovalbumin solutions as determined by UVabsorbance spectroscopy Target Concentration Actual Concentration(mg/mL) (mg/mL) 0.4 0.371 ± 0.001 1.5 1.374 ± 0.003 2.5 2.236 ± 0.040

TABLE 5 Nanocluster sizes Mode Geometric Mean Aerodynamic Targetconcentration of Diameter* Diameter ovalbumin (mg/mL) (μm) (μm) 0.4 6.252.384 ± 1.775 1.5 5.15 2.468 ± 1.931 2.5 5.10 2.447 ± 1.918 *See FIG. 8for size distribution.

Scanning electron microscopy (SEM): The size and morphology of thenanoclusters were evaluated using a LEO 1550 field emission scanningelectron microscope with secondary electron detection. The nanoclusterswere coated on a platform and sputtered with gold prior to imaging at4000 and 10,000 times magnification. FIG. 9 includes SEM images of thenanoclusters comprising DOTAP/PLGA nanoparticles and ovalbumin.

Example 6 Assessment of Dry Powder Performance In Vitro

A multi-stage liquid impactor (MSLI) fitted with a mouthpiece and throatassembly can be used to evaluate the deposition performance of variousparticle formulations administered from a dry powder inhaler. Foradministration through a dry powder inhaler (DPI) such as theSpinhaler®. Or Rotahaler®, particles are first encapsulated in a large,two-piece gelatin capsule. The capsule is placed into a smallcompartment in the DPI, which is then twisted to either separate orrupture the capsule immediately prior to breath actuation. Since nopropellants or compressed gases are used for these DPIs, the breathingforce of the patient, or in our case the volumetric flow rate throughthe MSLI, disperses the powder.

Using this experimental set-up, several important performance parameterscan be evaluated, including the respirable fraction of a particleformulation, the mass depositing in the mouthpiece and throat assemblyand the fractions of particles depositing at different stages throughoutthe MSLI (assessed by removing each section and weighing the collectedparticle mass). Particle batches depositing with high efficiency to thelower stages (˜1-5 μm cut-off) of the MSLI will be deemed as “deep lung”formulations suitable for ciprofloxacin encapsulation experiments.

Example 7 Identification of Nanocluster Formulations that can Entrap,Deposit, and Release Ciprofloxacin

Nanoclusters can be formulated for controlled release of ciprofloxacinfor ˜1 week. A complete analysis of nanocluster physicochemicalproperties, dispersion and release of the drug can be prepared by themethods described throughout this specification. The nanoclusters, inone embodiment, can be made with nanoparticles of pure ciprofloxacin orciprofloxacin encapsulated in PLGA nanoparticles. Ciprofloxacin is abroad spectrum antibiotic, especially effective against gram negativebacteria.

Nanocluster dispersability and ciprofloxacin release kinetics:Nanocluster formulations can be reformulated to determine controlledrelease of ciprofloxacin, taking care to maintain the same fabricationprocedure and resulting structure designed for deep lung deposition.Ciprofloxacin (Sigma, Inc.) can be encapsulated by co-dissolving withthe polymer phase and will be partially suspended in the polymer phaseor dissolved in a co-solvent if low solubility in the polymer phase isan issue. Dissolution studies ascertain the release kinetics ofciprofloxacin. These studies are performed in phosphate buffered salinesolution (pH 7.4) at physiological temperature (37° C.). Approximately10-20 mg of each particle formulation is placed in 2 mL microcentrifugetubes shaken at 150 rpm. Release samples will be tested byintermittently centrifuging samples to separate nanoparticles (15,000rpm), collecting 1-1.5 mL of supernatant, replacing supernatant withfresh buffer and resuspending the samples. The supernatant will then beanalyzed by spectrophotometry at ˜350 nm to determine the concentrationof ciprofloxacin at each time point while avoiding detection of polymerdissolution products. The release of ciprofloxacin from the variousnanocluster formulations will be conducted in triplicate and the averageand standard deviation is calculated. The initial loading ofciprofloxacin in nanocluster formulations is determined by dissolving˜10 mg of each formulation in triplicate in dimethylsulfoxide andmeasuring the absorbance at ˜350 nm. Absorbance values for formulationsof nanoclusters without ciprofloxacin are used as blanks. The calculatedamount of ciprofloxacin per mass of polymer is termed the drug loading.This number can be divided by the mass of ciprofloxacin per mass ofpolymer entered into the experiment to calculate the drug encapsulationefficiency. The summed mass of ciprofloxacin released over time is thendivided by the drug loading to arrive at the cumulative percentreleased. Analogous samples of nanoclusters can be prepared to determinethe dispersion kinetics based on measuring the turbidity of the samplesolution at 480 nm.

Reformulation and optimization of controlled release: Generating a nearconstant release of ciprofloxacin for ˜1 week may include reformulationof nanoclusters. If drug “bursting” (rapid initial release) occurs orincreased duration of release is desired, higher molecular weight PLGAor PLGA with a higher lactide content will be used as each of theseprolong degradation of the polymer phase. In addition, increasing thesize of constituent nanoparticles to decrease the rate of ciprofloxacinrelease can be used. The maintenance of small (e.g., <200 nm)nanoparticles can be used as a way to avoid phagocytosis or otherclearance mechanisms from the lung epithelium.

Example 8 Paclitaxel Nano-Agglomerates as Dry Powder for PulmonaryDelivery

Paclitaxel (PX), L-a-phosphatidylcholine (lecithin; Lec), cetyl alcohol(CA), L-leucine (Leu), polyvinylpyrrolidone (PVP K90, Mw˜36,000) andsodium chloride were purchased from Sigma Chemicals Co, U.S.A. PluronicF-127 (PL, Mw˜12,220) was purchased from BASF, The Chemical Company,U.S.A. Polyvinyl alcohol (PVA; Mw=22,000, 88% hydrolyzed) was purchasedfrom Acros Organics, N.J., U.S.A. Potassium dihydrogen phosphate,disodium hydrogen phosphate, acetone, ethanol and acetonitrile werepurchased through Fisher Scientific. Floatable dialysis membrane units(Mw cut-off=10,000 Da) were obtained from Spectrum Laboratories Inc.,U.S.A. A549 cells were obtained from the American Type CultureCollection (ATCC, Rockville, Md.). The cell culture medium (Ham's F-12Nutrient Mixture, Kaighn's modified with L-glutamine) was purchasedthrough Fisher Scientific. Fetal bovine serum (FBS) was purchased fromHyclone. Penicillin-streptomycin was purchased from MB Biomedical, LLC.Trypsin-EDTA was purchased through Gibco. MTS reagent [tetrazoliumcompound;3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt] was purchased from Promega, U.S.A. Double-distilled waterwas used throughout the study, provided by an EASYpure® RODI (BarnsteadInternational, Model # D13321).

Fabrication and Characterization of Paclitaxel Nanoparticles.

Nanosuspensions were prepared using a precipitation technique. The drugwas precipitated by direct injection of acetone solution of paclitaxel,0.1% w/v, in waters at a rate of 1 mL/min under sonication (FisherScientific, Sonic Dismembrator) with an amplitude of 46%. The chosensurfactants for the study included hydrophobic (cetyl alcohol),hydrophilic (PL, PVA and PVP K90) and amphoteric (lecithin). Thehydrophobic and amphoteric surfactants were added to the drug organicsolvent solution and the contents were allowed to stand at roomtemperature for 30 to 45 minutes with occasional vortexing to allowcomplete solubilization of the drug and the surfactants. Hydrophilicsurfactants were added to the aqueous phase. Surfactants were usedindividually or in combination as reported.

The particle size and zeta potential of the nanosuspensions weredetermined by dynamic light scattering (Brookhaven, ZetaPALS). Zetapotential measurements were performed using 1 mM KCl solution. Allmeasurements were performed in triplicate.

Preparation of Paclitaxel Nanoparticle Agglomerates

The paclitaxel nanoparticle agglomerates were prepared by addition ofL-leucine powder to agglomerate nanoparticle suspensions followed byhomogenization at 25,000 rpm for 30 sec. The amount of L-leucine addedwas adjusted to a drug:leucine ratio equal to 1:1. The size ofpaclitaxel nanoparticle agglomerates was measured in Isoton diluentusing a Coulter Multisizer 3 (Beckman Coulter Inc.) equipped with a 100mm aperture after three hours of incubation with the agglomeratingagent. The suspensions were kept overnight at room temperature to allowevaporation of acetone and then frozen at −80° C. and transferred to afreeze dryer (Labconco, FreeZone 1). Drying lasted for 36 hours toremove all appreciable water content. Lyophilized powder was stored atroom temperature for further characterization.

Characterization of the Prepared Nanoparticle Agglomerates

Determination of Particle Size Distribution

The particle size of the dispersed nanoparticle agglomerates as well asthe resuspended lyophilized powder was measured using a CoulterMultisizer 3. The particle size distributions are shown in FIG. 10.

Flowability Characteristics

The flow properties of the nanoparticle agglomerates were assessed byangle of repose (tan θ=height/radius) measurement of the dried powders.The fixed-height cone method was used. A glass funnel with cut stemsurface of 5 mm internal diameter was fixed at 2.5 cm height over a flatsurface. The powders were allowed to flow gently through the funneluntil a cone was formed and reached the funnel orifice. The flow ofpowder was then stopped and the average diameter of the formed cone (D)was measured. The area of the base of the cone was taken as a measure ofthe internal friction between the particles. The angle of repose wascalculated by the equation: tan θ=height/radius.

In addition, the bulk density, Hausner ratio (Tapped density/bulkdensity) and Can's index (Ci) [(Tapped density−bulk density)/Tappeddensity×100%] were also determined for the dried powders. Ten mg ofpowders were weighed and poured into a 10 mL graduated measuringcylinder. The bulk volume occupied (Vb) was recorded. The measuringcylinder was tapped until a constant value was obtained and the tappedvolume was recorded (Vt). The process was repeated at least three timesand the average was taken in each case. The bulk and tapped densities ofpowder were calculated by dividing the weight by the corresponding bulkvolume or tapped volume recorded.

Measurement of Aerodynamic Diameter

The aerodynamic size distributions of the agglomerate powders weremeasured directly from lyophilized powder by time-of-flight measurementusing an Aerosizer LD (Amherst Instruments) equipped with a 700 mmaperture operating at 6 psi.

The theoretical mass-mean aerodynamic diameter (d_(aero)) of thenanoparticle agglomerates was determined from the geometric particlesize and tapped density using the following relationship:

$d_{aero} = {\frac{d_{geo}}{\gamma}\sqrt{\rho/\rho_{a}}}$

Where d_(geo)=geometric diameter, γ=shape factor (for a sphericalparticle, γ=1; for aerodynamic diameter calculations, the particles inthis study were assumed to be spherical), ρ=particle bulk density andρ_(a)=water mass density (1 g/cm³). Aerodynamic size distributions ofpaclitaxel nanoparticle agglomerates is shown in FIG. 11. Tapped densitymeasurements underestimate particle bulk densities since the volume ofparticles measured includes the interstitial space between theparticles. The true particle density, and therefore the aerodynamicdiameter of a given powder, is expected to be slightly larger thanreported.

Aerosolization Performance of Nano-Agglomerate Dry Powders

Aerodynamic characteristics of selected nanoparticle agglomerates werestudied in vitro using a Tisch Ambient Cascade Impactor (TischEnvironmental, Inc., Ohio). The study was carried out by applying ˜20 mgpowder manually into the orifice of the instrument at an air flow rateof ˜30 L/min. Cut-off particle aerodynamic diameters for each stage ofthe impactor were: pre-separator (10.00 mm), stage 0 (9.00 mm), stage 1(5.8 mm), stage 2 (4.7 mm), stage 3 (3.3 mm), stage 4 (2.1 mm), stage 5(1.1 mm), stage 6 (0.7 mm), stage 7 (0.4 mm) and filter (0 mm).Nanoparticle agglomerates deposited on each stage of the impactor weredetermined by measuring the difference in weight of filters placed onthe stages. The mass median aerodynamic diameter, MMAD, and geometricstandard deviation, GSD, were obtained by a linear fit of the cumulativepercent less-than the particle size range by weight plotted on aprobability scale as a function of the logarithm of the effectivecut-off diameter. FIG. 12 shows the distribution of Paclitaxel powder asreceived and nanoparticle agglomerate formulations deposited on thestages of a cascade impactor at a flow rate of ˜30 L/min.

Imaging of Particles by Transmission Electron Microscopy

Image data was used to corroborate the size of nanoparticles andnanoparticle agglomerates and to observe their morphological aspects.Transmission electron micrographs (TEM) were obtained for paclitaxelnanoparticles and nanoparticle agglomerates using a JEOL 1200 EXIItransmission electron microscope. Initially, carbon-coated grids(Electron Microscopy Sciences) were floated on a droplet of thesuspensions on a flexible plastic film (Parafilm), to permit theadsorption of the particles onto the grid. After this, the grid wasblotted with a filter paper and air dried for 1 hr.

Determination of Process Yield and Loading Efficiency

The lyophilized powder for the prepared nanoparticle agglomerates wasweighed and the yield was calculated using the following expression:

Paclitaxel loading efficiency was assessed by dispersing one mg of thelyophilized powder in 10 mL ethanol. The dispersion was sonicated in abath-type sonicator (Branson 3510) for 30 min. Then the solution wascentrifuged (Beckman, Avanti TM) at—15,000 rpm for 30 min to removeinsoluble ingredients and the amount of drug in the supernatant wasdetermined spectrophotomerically (Agilent C) at 228 nm.

Dissolution Studies

The dissolution of the prepared nanoparticles and nanoparticleagglomerates was determined and compared with the dissolutioncharacteristics of the drug powder as received. The dissolution ofpaclitaxel was carried out at 37±0.5° C. in a 1 liter beaker. A knownamount (˜10 mg) of the lyophilized powder was suspended in 10 mLphosphate buffered saline (PBS, pH 7.4) and was placed into a floatabledialysis membrane unit (Mw cut-off=10,000 Da), and the unit was allowedto float in a 500 mL of PBS. The solution was stirred at a constantspeed (100 rpm) using a magnetic stirrer (Barnstead, ThermolyneMIRAKTM). At predetermined time intervals for a total period of 8 hours,serial samples (1 mL) of the medium were withdrawn from the dialysis bagand centrifuged for 30 minutes at ˜13,000 rpm. The nanoparticles-freesupernatant was removed and extracted with 3 mL of ethanol. The ethanolextract was analyzed for paclitaxel concentration using a reverse-phaseHPLC method. Studies were conducted in triplicate. A Shimadzu HPLCsystem including a solvent delivery pump (Shimadzu LC-10AT), acontroller (Shimadzu SCL-10A), an autoinjector (Shimadzu SIL-10AxL), anda UV detector (Shimadzu SPD-10A) was used in this study. The peak areaswere integrated using Shimadzu Class VP (Version 4.3). A 4.6 mm×100 mmlong Zorbax SB C-8 column (Agilent C) with a particle diameter of 3.5 μmwas used. During the assay, paclitaxel was eluted isocratically at amobile phase flow rate of 0.9 mL/minute and monitored with a UV detectoroperating at 228 nm. The mobile phase for the assay consisted of anacetonitrile and water mixture (50:50 v/v). The run time for the assaywas 20 minutes, and the retention time for paclitaxel was 10.7 minutes.FIG. 13 shows the in-vitro dissolution profiles of paclitaxel in PBS (pH7.4) from pure paclitaxel powder and two different nanoparticle (NP) andnanoparticle agglomerate formulations (NA).

Cytotoxicity Assay

The cytotoxicity of selected nanoparticles and nanoparticle agglomerateswas assessed using the CellTiter 96® Aqueous Cell Proliferation Assay(Promega) and compared with paclitaxel powder as received, lecithin, PVPK90, L-leucine, physical mixtures of these ingredients and blanknanoparticle agglomerates. In this experiment, 8×104 A549 cells/wellwere seeded in 96-well microtiter plates. At the end of the incubationperiod (12 h), 20 ml of MTS reagent solution was added to each well andincubated for 3 h at 37° C. The absorbance was measured at 490 nm usinga microtiter plate reader (SpectraMax, M25, Molecular Devices Corp.,CA). The percentage of viable cells with all tested concentrations wascalculated relative to untreated cells. FIG. 14 shows the viability ofA549 cells in the presence of formulation components as determined by anMTS assay.

TABLE 6 IC50 for Paclitaxel Formulations Formulation IC₅₀ Purepaclitaxel powder 1.8 mg/ml F1NP 2.1 mg/ml F1NA 1.9 mg/ml PM1 1.5 mg/mlBlank1 0.88 mg/ml  F2NP 1.6 mg/ml F2NA 2.1 mg/ml PM2 1.5 mg/ml Blank21.8 mg/ml

TABLE 7 Paclitaxel formulations used in the studies. Cetyl PaclitaxelLecithin PVP K90 alcohol Formulation (% w/v) (% w/v) (% w/v) (% w/v) F10.1 0.02 0.01 F2 0.1 0.02 F3 0.1 0.02 0.01

TABLE 8 Physical properties of Paclitaxel nanoparticles (values =average ± standard deviation). Nanoparticle size Formulation (nm)Zeta-potential (mV) Polydispersity F1^(a) 298.7 ± 10.3 25.1 ± 0.7  0.04± 0.03 F2^(b) 339.1 ± 13.6 24.7 ± 1.5 0.18 ± 0.1 F3^(c) 358.5 ± 8.1  22.4 ± 1.02 0.31 ± 0.1 ^(a)F1 = 0.1:0.02:0.01; PX:Lec:PVP K90 ^(b)F2 =0.1:0.02; PX:Lec ^(c)F3 = 0.1:0.02:0.01; PX:Lec:CA

TABLE 9 Characteristics of Paclitaxel nanoparticle agglomerates (values= average ± standard deviation.). Formulations Characteristics F1^(a)F2^(b) Geometric particle size (μm) of NA^(c) before 2.8 ± 0.5 3.7 ± 0.9lyophilization Geometric particle size (μm) of lyophilized 4.8 ± 1.3 5.4± 1.6 NA^(c) MMAD_(A) ^(d) of lyophilized NA^(c)  1.5 ± 0.08 1.7 ± 0.4^(a)F1 = 0.1:0.02:0.01; PX:Lec:PVP K90 ^(b)F2 = 0.1:0.02; PX:Lec ^(c)NA:Nanoparticle agglomerates. ^(d)MMAD: Mass median aerodynamic diameterobtained from Aerosizer.

TABLE 10 Yield, loading and dissolution behavior of Paclitaxelnanoparticle agglomerates (values = average ± standard deviation.).Formulations Characteristics F1^(a) F2^(b) % Process yield oflyophilized NA^(c)    85.9 ± 2.7 89.8 ± 3.1 Drug loading of lyophilizedNA^(c)    85.1 ± 3.9 85.9 ± 8.7 Q_(8h) ^(e)NP^(d) 66.4% ± 2.4  60.7% ±13.04 Q_(8h) ^(e) NA^(c) 43.6% ± 4.3 40.4% ± 2.9   ^(a)F1 =0.1:0.02:0.01; PX:Lec:PVP K90 ^(b)F2 = 0.1:0.02; PX:Lec ^(c)NA:Nanoparticle agglomerates. ^(d)NP: Nanoparticles. ^(e)Q_(8h): %Paclitaxel dissolved after 8 hours.

TABLE 11 Cascade impaction results of lyophilized paclitaxelnanoparticle agglomerates (values = average ± standard deviation.).Formulations Characteristics of the Paclitaxel as lyophilized NA^(c)F1^(a) F2^(b) received At flow % EF^(d) 71.9 ± 4.9  71.6 ± 15.4 68.3 ±6.1 rate of % RF^(e) <5.7 95.7 ± 3.01 95.3 ± 4.1  0.39 ± 0.3 ~30 <3.383.8 ± 2.8  79.55 ± 4.8  0 L//min MMAD^(f) 1.5 ± 0.1 1.9 ± 0.4  0.08 ±0.02 GSD^(g) 2.26 ± 0.09 2.29 ± 0.05  2.2 ± 0.03 ^(a)F1 = 0.1:0.02:0.01;Px:Lec:PVP K90 ^(b)F2 = 0.1:0.02; Px:Lec ^(c)NA: Nanoparticleagglomerates. ^(d)% EF: Percent emitted fraction. ^(e)RF: Percentrespirable fraction. ^(f)MMAD: Mass median aerodynamic diameter.^(g)GSD: Geometric standard deviation.

Example 9 Application of Budesonide Nanoparticles

Budesonide is a potent nonhalogenated corticosteroid with highglucocorticoid receptor affinity, airway selectivity and prolongedtissue retention. It inhibits inflammatory symptoms, such as edema andvascular hyperpermeability. Budesonide is already applied through drypowder inhalers (DPI, Pulmicort), metered dose inhalers (pMDI,Rhinocort) or ileal-release capsules (Entocort). This drug is consideredone of the most valuable therapeutic agents for the prophylactictreatment of asthma despite its poor solubility in water (21.5 mg/mlunder constant agitation).

This example demonstrates the translation of budesonide nanosuspensionsinto dry powder formulations capable of effective deposition and rapiddissolution. Different surfactants were used to create surface charge onthe nanoparticles and charge interactions were leveraged to agglomeratenanoparticles into nanoparticle agglomerates exhibiting a particle sizerange of ˜2-4 μm. Nanoparticle suspensions were evaluated by measuringparticle size, polydispersity and zeta potential. Nanosuspensions werethen agglomerated and lyophilized to obtain dry powders composed ofmicron-sized agglomerates. Nanoparticle agglomerates were characterizedby the determination of particle size, aerolization efficiencies,flowability characteristics, process yield and loading efficiency.Finally, dissolution studies were performed for the selectednanoparticles and nanoparticle agglomerates, which were compared withthe stock drug.

Materials and Methods

Budesonide (Bud), L-a-phosphatidylcholine (lecithin; Lec), cetyl alcohol(CA), L-leucine (Leu), polyvinylpyrrolidone (PVP), sorbitan tri-oleate(Span 85) and sodium chloride were purchased from Sigma Chemicals Co,U.S.A. Pluronic F-127 (PL, Mw˜12,220) was purchased from BASF, TheChemical Company, U.S.A. Polyvinyl alcohol (PVA; Mw=22,000, 88%hydrolyzed) was purchased from Acros Organics, N.J., U.S.A. Potassiumdihydrogen phosphate, disodium hydrogen phosphate, acetone, ethanol andacetonitrile were purchased through Fisher Scientific. Floatabledialysis membrane units (Mw cut-off=10,000 Da) were obtained fromSpectrum Laboratories Inc., U.S.A. A549 cells were obtained from theAmerican Type Culture Collection (ATCC, Rockville, Md.). The cellculture medium (Ham's F-12 Nutrient Mixture, Kaighn's modified withL-glutamine) was purchased through Fisher Scientific. Fetal bovine serum(FBS) was purchased from Hyclone. Penicillin-streptomycin was purchasedfrom MB Biomedical, LLC. Trypsin-EDTA was purchased through Gibco. MTSreagent [tetrazolium compound;3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt] was purchased from Promega, U.S.A. Double-distilled waterwas used throughout the study, provided by an EASYpure® RODI (BarnsteadInternational, Model # D13321).

Preparation of Budesonide Nanosuspensions

Nanosuspensions were prepared using a precipitation technique. Briefly,solutions of budesonide in acetone were prepared at concentrations of0.1 and 0.2% w/v and water was used as nonsolvent. To precipitate thedrug, the solution of budesonide was directly injected into thenon-solvent at a rate of 1 mL/min under sonication (Fisher Scientific,Sonic Dismembrator) with an amplitude of 46% in an ice bath. Theselected surfactants for the study included hydrophobic (cetyl alcoholand Span 85), hydrophilic (PL, PVA and PVP) and amphoteric (lecithin).The hydrophobic and amphoteric surfactants were added to the drugorganic solvent solution and the contents were allowed to stand at roomtemperature for 30 to 45 minutes with occasional vortexing to allowcomplete solubilization of the drug and the surfactants. Hydrophilicsurfactants were added to the aqueous phase. Surfactants were usedindividually or in combination as reported.

Agglomeration of Budesonide Nanoparticles

The budesonide nanoparticle agglomerates were prepared by slow additionof L-leucine solution (1% w/v) in water to agglomerate nanoparticlesuspensions during homogenization at 25,000 rpm for 30 sec. The amountof L-leucine added was adjusted to a drug:leucine ratio equal to 1:1.The size of budesonide nanoparticle agglomerates was measured in Isotondiluent using a Coulter Multisizer 3 (Beckman Coulter Inc.) equippedwith a 100 μm aperture after three hours of incubation with theagglomerating agent. The agglomerated suspensions were kept overnight atroom temperature to allow evaporation of acetone and then frozen at −80°C. and transferred to a freeze dryer (Labconco, FreeZone 1). Dryinglasted for 36 hours to remove all appreciable water content. Lyophilizedpowder was stored at room temperature for further characterization.

Characterization of the prepared nanoparticles and nanoparticleagglomerates.

Particle Size Analysis and Zeta Potential Measurement of the SelectedNanosuspensions.

The size and zeta potential of the nanosuspensions were determined bydynamic light scattering (Brookhaven, ZetaPALS). Zeta potentialmeasurements were performed using 1 mM KCl solution. All measurementswere performed in triplicate.

Determination of Particle Size Distribution and Aerodynamic Diameter ofthe Prepared Nanoparticle Agglomerates.

The particle size of the dispersed nanoparticle agglomerates as well asthe resuspended lyophilized powder was measured using a CoulterMultisizer 3. The aerodynamic size distributions of the agglomeratepowders were measured directly from lyophilized powder by time-of-flightmeasurement using an Aerosizer LD (Amherst Instruments) equipped with a700 μm aperture operating at 6 psi.

Aerosolization of Nanoparticle Agglomerates

Aerodynamic characteristics of selected nanoparticle agglomerates werestudied in vitro using a Tisch Ambient Cascade Impactor (TischEnvironmental, Inc., Ohio). The study was carried out by applying ˜20 mgpowder manually into the orifice of the instrument at three air flowrates; ˜15 L/min, ˜30 L/min and ˜60 L/min. Cut-off particle aerodynamicdiameters at 30 L/min for each stage of the impactor were: pre-separator(10.00 μm), stage 0 (9.00 μm), stage 1 (5.8 μm), stage 2 (4.7 μm), stage3 (3.3 μm), stage 4 (2.1 μm), stage 5 (1.1 μm), stage 6 (0.7 μm), stage7 (0.4 μm) and filter (0 μm). Nanoparticle agglomerates deposited oneach stage of the impactor were determined by measuring the differencein weight of filters placed on the stages. The mass median aerodynamicdiameter, MMAD, and geometric standard deviation, GSD, were obtained bya linear fit of the cumulative percent less-than the particle size rangeby weight plotted on a probability scale as a function of the logarithmof the effective cut-off diameter.

Transmission Electron Microscopy (TEM)

Image data was used to corroborate the size of nanoparticles andnanoparticle agglomerates and to observe their morphological aspects.Transmission electron micrographs (TEM) were obtained for budesonidenanoparticles and nanoparticle agglomerates using a JEOL 1200 EXIItransmission electron microscope. Initially, carbon-coated grids(Electron Microscopy Sciences) were floated on a droplet of thesuspensions on a flexible plastic film (Parafilm), to permit theadsorption of the particles onto the grid. After this, the grid wasblotted with a filter paper and air dried for 1 hr.

SSNMR Analysis

All 13C spectra were collected using a Chemagnetics CMX-300 spectrometerusing ramped amplitude cross-polarization (RAMP), magic-angle spinning(MAS), and SPINAL-64 decoupling. Samples were packed in 7 mm zirconiarotors using Teflon® end caps, and spun at 4 kHz in a 7 mm spin modulefrom Revolution NMR.

All spectra are the sum of 2,000-48,000 transients collected using a1-1.5 sec pulse delay, a contact time of 0.5-2 ms, and a 1H 90° pulsewidth of 3-4.5 μs. The free induction decays consisted of 512-2048points with a dwell time of 33.3 μs. The spectra were externallyreferenced to tetramethylsilane using the methyl peak of3-methylglutaric acid at 18.84 ppm.

The assignment of the peaks in the ¹³C spectrum of the “as received”budesonide was performed using the modified spectral editing methods ofHu et al. and the ¹³C solution predictions from ChemBioDraw Ultra(version 11.0) from CambridgeSoft and ACD/CNMR Predictor (version 7.09)from ACD/Labs. The spectral editing subspectra were collected using theparameters given in Table 12. These parameters were optimized using3-methylglutaric acid.

TABLE 12 Parameters used to collect ¹³C spectral editing subspectra.*Pulse Number of Subspectra Sequence t_(CP) (μs) t_(PI) (μs) t_(DD) (μs)t_(SL) (μs) Transients All (+) 1500 0.2 — 133 800 C + CH₃ (+) 1500 0.2130 3 800 C (−) 400 135 130 3 8,000 CH (+) 40 24 — 133 6,000 (−) 37 0.2130 1 6,000 CH₂ (+) 80 39 — 133 6,000 (−) 78 0.2 147 1 6,000 *= Allsubspectra were collected with a 1.5 sec pulse delay, a ¹H 90° pulsewidth of 3.1 μs, 1024 acquisition points with a dwell time of 33.3 μs,and a magic-angle spinning rate of 4 kHz.

Determination of Process Yield

The lyophilized powder for the prepared nanoparticle agglomerates wasweighed and the yield was calculated using the following expression:

${\%\mspace{14mu}{Process}\mspace{14mu}{yield}} = {\frac{{Recovered}\mspace{14mu}{mass}}{{Mass}\mspace{14mu}{entered}\mspace{14mu}{into}\mspace{14mu}{the}\mspace{14mu}{experiment}} \times 100}$

Budesonide Loading Efficiency Measurement

Budesonide loading efficiency was assessed by dispersing one mg of thelyophilized powder in 10 mL ethanol. The dispersion was sonicated in abath-type sonicator (Branson 3510) for 2 hours and then kept overnightat room temperature to allow complete dissolution of the drug byethanol. Then the solution was centrifuged (Beckman, Avanti TM) at˜15,000 rpm for 30 min to remove insoluble surfactants and L-leucine andthe amount of drug in the supernatant was determinedspectrophotometrically (Agilent C) at 243 nm. Drug loading was definedas follows:

Flowability Characteristics

The flow properties of the nanoparticle agglomerates were assessed byangle of repose (tan θ=height/radius) measurement of the dried powders.The fixed-height cone method was used. A glass funnel with cut stemsurface of 5 mm internal diameter was fixed at 2.5 cm height over a flatsurface. The powders were allowed to flow gently through the funneluntil a cone was formed and reached the funnel orifice. The flow ofpowder was then stopped and the average diameter of the formed cone (D)was measured. The area of the base of the cone was taken as a measure ofthe internal friction between the particles. The angle of repose wascalculated by the equation: tan θ=height/radius.

In addition, the bulk density, Hausner ratio (Tapped density/bulkdensity) and Carr's index (Ci) [(Tapped density−bulk density)/Tappeddensity×100%] were also determined for the dried powders. Five mg ofpowders were weighed and poured into a 10 mL graduated measuringcylinder. The bulk volume occupied (V_(b)) was recorded. The measuringcylinder was tapped until a constant value was obtained and the tappedvolume was recorded (V_(t)). The process was repeated at least threetimes and the average was taken in each case. The bulk and tappeddensities of powder were calculated by dividing the weight by thecorresponding bulk volume or tapped volume recorded.

Dissolution Studies

The dissolution of the prepared nanoparticles and nanoparticleagglomerates was determined and compared with the dissolutioncharacteristics of the stock drug. The dissolution of budesonide wascarried out at 37±0.5° C. in a 400 mL beaker. A known amount (˜10 mg) ofthe lyophilized powder was suspended in 10 mL phosphate buffered saline(PBS, pH 7.4) and was placed into a floatable dialysis membrane unit (Mwcut-off=10,000 Da), and the unit was allowed to float in a beakercontaining 300 mL of PBS. The solution was stirred at a constant speed(100 rpm) using a magnetic stirrer (Barnstead, Thermolyne MIRAKTM). Atpredetermined time intervals for a total period of 8 hours, aliquots (5ml) of the medium were removed and fresh medium was immediately added tocontinue the dissolution study. Studies were conducted in triplicate.The budesonide concentration was analyzed using a reverse-phase HPLCmethod. A Shimadzu HPLC system including a solvent delivery pump(Shimadzu LC-10AT), a controller (Shimadzu SCL-10A), an autoinjector(Shimadzu SIL-10AxL), and a UV detector (Shimadzu SPD-10A) was used inthis study. The peak areas were integrated using Shimadzu Class VP(Version 4.3). A 4.6 mm×100 mm long Zorbax SB C-18 column (Agilent C)with a particle diameter of 3.5 μm was used. During the assay,budesonide was eluted isocratically at a mobile phase flow rate of 0.6mL/minute and monitored with a UV detector operating at 254 nm. Themobile phase for the assay consisted of an acetonitrile and watermixture (45:55 v/v). The run time for the assay was 20 minutes, and theretention time for budesonide was 14.01 minutes.

Cytotoxicity Assay

The cytotoxicity of selected nanoparticles and nanoparticle agglomerateswas assessed using the CellTiter 96® Aqueous Cell Proliferation Assay(Promega) and compared with stock budesonide, lecithin, leucine,physical mixtures of these ingredients and blank nanoparticleagglomerates. In this experiment, 8×104 A549 cells/well were seeded in96-well microtiter plates. At the end of the incubation period (12 h),20 ml of MTS reagent solution was added to each well and incubated for 3hours at 37° C. The absorbance was measured at 490 nm using a microtiterplate reader (SpectraMax, M25, Molecular Devices Corp., CA). Thepercentage of viable cells with all tested concentrations was calculatedrelative to untreated cells.

Results and Discussion

Fabrication of Budesonide Nanoparticles

Various methods have been reported for generating nanoparticles ofpoorly water soluble drugs. A precipitation method was selected toproduce budesonide nanoparticles. Different concentrations of the drugand various types and ratios of surfactants, individually or incombination, were evaluated as a means to control the particle size andsurface charge. Surfactants were chosen from excipients regarded assuitable for inhalation that have been designated as safe for human use.Formulations prepared using PVP and PVA in different ratios producedvery large particle sizes even when combined with other surfactants. Themean particle size of formulations containing lecithin, cetyl alcohol,Span 85 and/or PL ranged from ˜130 to 323 nm. Formulations containingSpan 85 alone or in combination with lecithin yielded the smallestparticle size but since Span is liquid at room temperature, it was notsuitable for use in dry powder formulations.

Attempts to generate budesonide nanoparticles using PL alone or incombination with lecithin yielded reasonable particle sizes (˜129-270nm) but offered very low nanoparticle yields and high polydispersityvalues. Selected surfactant combinations for preparing budesonidenanosuspensions in acetone were designated F1 (0.1% w/v Bud+0.02% w/vLec), F2 (0.1% w/v Bud+0.02% w/v CA+0.01% w/v PL) and F3 (0.2% w/vBud+0.04% w/v CA+0.02% w/v PL) as reported in Table 13. These surfactantcombinations demonstrated small particle size and could be used in drypowder formulations. A small change in zeta potential was observed withdifferent types of surfactants and the values ranged from 22.5-25.1 mV(Table 14). The charged surface of the nanoparticles provided thepotential for destabilizing this colloid via interaction with aagglomerating agent to form nanoparticle agglomerates.

TABLE 13 Composition of the selected formulations. Budesonide LecithinCetyl alcohol Pluronic F127 Formulation (% w/v) (% w/v) (% w/v) (% w/v)F1 0.1 0.02 F2 0.1 0.02 0.01 F3 0.2 0.04 0.02

TABLE 14 Physical properties of budesonide nanoparticle (values =average ± standard deviation.). Nanoparticle size Formulation (nm)Zeta-potential (mV) Polydispersity F1^(a) 160.9 ± 15.6 25.1 ± 1.3 0.41 ±0.1  F2^(b) 188.8 ± 26.3 24.2 ± 1.1 0.34 ± 0.02 F3^(c) 232.2 ± 11.2 22.5± 0.5 0.33 ± 0.02 ^(a)F1 = 0.1:0.02; Bud:Lec ^(b)F2 = 0.1:0.02:0.01;Bud:CA:PL ^(c)F3 = 0.2:0.04:0.02; Bud:CA:PL

Agglomerated Budesonide Nanoparticles Yielded Desirable AerosolCharacteristics

The mechanism to control nanoparticle agglomeration is mainly driven byleveraging the competitive processes of attraction (van der Waals force)and repulsion (electrostatic repulsive force or steric hindrance barrieror both). If particles are mainly stabilized electrostatically,disruption of the electrostatic double layer surrounding the particleswill result in the agglomeration of nanoparticles. The addition ofagglomerating agents has also been speculated to decreases the cohesionbetween particles. It is thought that these agents may interfere withweak bonding forces between small particles, such as van der Waals andCoulomb forces. These agents may act as weak links or “chain breakers”between the particles which are susceptible to disruption in theturbulent airstream created during inhalation. The amino acid,L-leucine, used as a agglomerating agent in these studies may also actas an anti-adherent material to yield a high respirable fraction of theagglomerated budesonide nanoparticles.

Agglomeration of nanoparticles resulted in the formation of agglomerateswithin the micrometer or sub-micrometer scale consisting ofclosely-packed nanoparticles. Nanoparticle agglomerates were preparedthrough the slow incorporation of a agglomerating agent (L-leucine)during homogenization (25,000 rpm) for 30 sec. The geometric sizedistribution of the prepared nanoparticle agglomerates was measured inIsoton diluent using a Coulter Multisizer 3. The size average of thethree selected nanoparticle agglomerate formulations ranged from ˜2-4 μm(Table 15). The size distributions of resuspended lyophilized powderswere slightly broader and the average particle size was slightlyincreased, when compared to the nanoparticle agglomerates prior tolyophilization (Table 15 and FIG. 15). This may be due to the depositionof nanoparticles on agglomerates during lyophilization or to cohesionbetween agglomerates as a result of drying. The key physical parameterthat predicts the site of aerosol deposition within the lungs forparticles larger than several hundred nanometers is the aerodynamicdiameter (d_(aero)). The aerodynamic diameter of the agglomeratednanoparticles, measured by an Aerosizer LD, was smaller than thegeometric diameter and the aerodynamic size distribution was narrowerthan the geometric size distribution (Table 15 and FIG. 16). Whencompared to the geometric diameter, the lower aerodynamic diameter waslikely due to the low density of nanoparticle agglomerates.

TABLE 15 Characteristics of budesonide nanoparticle agglomerates (values= average ± standard deviation.). Formulations Characteristics F1^(a)F2^(b) F3^(c) Geometric 2.8 ± 0.4 2.7 ± 0.4 3.2 ± 0.7 particle size (μm)of NA^(d) before lyophilization Geometric 3.1 ± 0.6 3.3 ± 0.7 3.9 ± 1.1particle size (μm) of lyophilized NA^(d) MMAD^(f) of 1.4 ± 1.7 2.1 ± 1.81.9 ± 1.8 lyophilized NA^(d) % Process 95.5 ± 4.9  92.7 ± 3.1  89.7 ±3.6  yield of lyophilized NA^(d) Loading 95.9 ± 3.6  86.5 ± 6.0  92.5 ±6.6  Efficiency of lyophilized NA^(d) Q_(8h) ^(g)NP^(e) 61.5% ± 1.6  75.5% ± 9.9   88.9% ± 3.0   Q_(8h) ^(g)NA^(d) 41.8% ± 4.6   51.2% ±5.1   63.1% ± 5.1   ^(a)F1 = 0.1:0.02; Bud:Lec ^(b)F2 = 0.1:0.02:0.01;Bud:CA:PL ^(c)F3 = 0.2:0.04:0.02; Bud:CA:PL ^(d)NA: Nanoparticleagglomerates. ^(e)NP: Nanoparticles. ^(f)MMAD: Mass median aerodynamicdiameter obtained from Aerosizer. ^(g)Q_(8 h): % budesonide dissolvedafter 8 hours.

The theoretical mass-mean aerodynamic diameters (daero) of thenanoparticle agglomerates, determined from the geometric particle sizeand tapped density, was found to be 2, 2.1 and 2.5 μm for F1, F2 and F3,respectively as calculated from the relationship:

$d_{aero} = {\frac{d_{geo}}{\gamma}\sqrt{\rho/\rho_{a}}}$

Where d_(geo)=geometric diameter, γ= shape factor (for a sphericalparticle, γ=1; for aerodynamic diameter calculations, the particles inthis study were assumed to be spherical), ρ= particle bulk density andρ_(a)= water mass density (1 g/cm³). Tapped density measurementsunderestimate particle bulk densities since the volume of particlesmeasured includes the interstitial space between the particles. The trueparticle density, and therefore the aerodynamic diameter of a givenpowder, is expected to be slightly larger than reported. Particles witha d_(aero) between 1 and 5 μm that are inhaled via the mouth are capableof efficient alveolar deposition, whereas d_(aero) between 4 and 10 μmare more likely to deposit primarily in the tracheobronchial region ofthe lungs. Therefore, the budesonide nanoparticle agglomerates withdaero in the 2-2.5 μm range are expected to deposit primarily in thealveolar region of the lungs.

Aerosizer results and theoretical MMAD calculations were corroborated bycascade impaction studies at air flow rates of ˜15 L/min, ˜30 L/min and˜60 L/min (FIG. 17). At these flow rates, most nanoparticle agglomerateswere deposited in stages 6 and 7 of the cascade impactor which wassuggestive of efficient aerosolization and a high fine particlefraction. The aerosolization efficiency of nanoparticle agglomerates wasrepresented by the percent emitted fraction (% EF), percent respirablefraction (RF), mass-median aerodynamic diameter (MMAD) and geometricalstandard deviation (GSD). The percent emitted fraction was determinedfrom the following equation:

${\%\mspace{14mu}{Emitted}\mspace{14mu}{fraction}\mspace{14mu}\left( {\%\mspace{14mu}{EF}} \right)} = {\frac{\begin{matrix}{{Total}\mspace{14mu}{particle}\mspace{14mu}{mass}\mspace{14mu}{collected}\mspace{14mu}{from}} \\{{the}\mspace{14mu}{stages}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{impactor}}\end{matrix}}{\begin{matrix}{{Total}\mspace{14mu}{particle}\mspace{14mu}{mass}\mspace{14mu}{entered}} \\{{into}\mspace{14mu}{the}\mspace{14mu}{impactor}}\end{matrix}\mspace{14mu}} \times 100}$

The high emitted fraction of nanoparticle agglomerates obtained at thetested flow rates suggested efficient aerosolization of the powder(Table 16). The percent respirable fraction (RF), referred to also asthe fine particle fraction of the total dose (FPFTD), was calculated asthe percentage of aerosolized particles that reached the lower sevenstages of the impactor (corresponding to aerodynamic diameters below 5.8μm), or the lower five stages (corresponding to aerodynamic diametersbelow 3.3 μm) according to the following equation:

${\%\mspace{14mu}{Respirable}\mspace{14mu}{fraction}\mspace{14mu}({RF})} = {\frac{\begin{matrix}{{Powder}\mspace{14mu}{mass}\mspace{14mu}{recovered}\mspace{14mu}{from}} \\{{terminal}\mspace{14mu}{stages}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{impactor}}\end{matrix}}{\begin{matrix}{{Total}\mspace{14mu}{particle}\mspace{14mu}{mass}} \\{{recovered}\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{impactor}}\end{matrix}} \times 100}$

The results of the respirable fraction also suggested the efficientaerosolization of nanoparticle agglomerate powders (Table 16). Thegeometric standard deviation (GSD) of the nanoparticle agglomerates wasdetermined from the following equation:

${G\; S\; D} = \left( \frac{d_{84.13\%}}{d_{15.87\%}} \right)^{1/2}$

Where d_(n) is the diameter at the nth percentile of the cumulativedistribution. The mass-mean geometric size of nanoparticle agglomeratesranged between 3 and 4 μm with a GSD of ˜2.3 μm (Table 16). Typical GSDvalues for aerosol particles are between 1.3-3.0.17 The mass-meanaerodynamic diameter (MMAD) of the selected nanoparticle agglomerates,as calculated from the cascade impaction results (Table 16) was close tothat obtained from the Aerosizer (Table 15) although it was slightlysmaller than the theoretical density values calculated from the tappeddensity indicating the suitability of the prepared nanoparticleagglomerate powders for peripheral lung deposition (i.e., <3 μm).

TABLE 16 Cascade impaction results of lyophilized budesonidenanoparticle agglomerates (values = average ± standard deviation).Characteristics of the Formulations lyophilized NA^(d) F1^(a) F2^(b)F3^(c) At flow % EF^(e) 74.9 ± 6.1 69.3 ± 5.5 86.5 ± 8.6 rate of % <5.796.3 ± 1.8 97.5 ± 1.3 97.9 ± 0.3 ~15 L//min RF^(f) <3.3 77.6 ± 3.9 81.2± 5.2 75.3 ± 1.7 MMAD^(g)  1.7 ± 0.1  1.5 ± 0.1  1.9 ± 0.3 GSD^(h)  2.4± 0.2  2.3 ± 0.1  2.3 ± 0.3 At flow % EF^(e) 75.3 ± 7.3 70.2 ± 4.1  81.2± 12.9 rate of % <5.7 97.1 ± 0.2 96.8 ± 1.5 96.9 ± 1.4 ~30 L//min RF^(f)<3.3 84.3 ± 3.9 87.7 ± 1.5 82.3 ± 4.2 MMAD^(g)  1.3 ± 0.2  1.2 ± 0.04 1.6 ± 0.3 GSD^(h)  2.3 ± 0.3  2.3 ± 0.4  2.3 ± 0.1 At flow % EF^(e)77.4 ± 6.6 72.2 ± 5.9  82.9 ± 12.7 rate of % <5.7 95.8 ± 0.2 95.9 ± 1.595.7 ± 1.7 ~60 L//min RF^(f) <3.3 87.1 ± 2.3 89.3 ± 2.5 83.6 ± 2.8MMAD^(g)  1.1 ± 0.2  1.1 ± 0.1  1.3 ± 0.2 GSD^(h)  2.3 ± 0.1  2.3 ± 0.2 2.3 ± 0.1 ^(a)F1 = 0.1:0.02; Bud:Lec ^(b)F2 = 0.1:0.02:0.01; Bud:CA:PL^(c)F3 = 0.2:0.04:0.02; Bud:CA:PL ^(d)NA: Nanoparticle agglomerates.^(e)% EF: Percent emitted fraction. ^(f)RF: Percent respirable fraction.^(g)MMAD: Mass median aerodynamic diameter. ^(h)GSD: Geometric standarddeviation.

Electron microscopy was used to study the morphology of budesonidenanoparticle and nanoparticle agglomerate formulations. Transmissionelectron micrographs (TEM) of F1 nanoparticles (FIG. 18A) depictedslightly elongated nanoparticles with smooth surfaces and a particlesize around 170 nm. TEM images of F1 nanoparticle agglomerates (FIG.18B) show that the nanoparticles were agglomerated into micron sizedagglomerates with irregular structure and some sharp edges.

FIG. 19 shows the ¹³C spectra of budesonide by itself and informulations. Both the budesonide as received and the leucine exhibitrelatively narrow lines (several tens of hertz), indicating that thesesamples are crystalline. Lecithin also had narrow lines, which isconsistent with it being a crystalline form of phosphatidylcholine;however, it is a semi-solid and therefore cannot be crystalline.Conversely the budesonide that was melt quenched had significantlybroader lines (several hundreds of hertz) indicating that the budesonideis consistent with it being amorphous. In the nanoparticles, the peaksfor budesonide are similar to the peaks in the budesonide that was meltquenched, although the peak at ˜180 ppm shows that there is a smallamount of crystalline budesonide in the nanoparticles. The tall, sharppeaks in the spectrum of the nanoparticle agglomerates align with thepeaks in the leucine spectrum and showed that the leucine in theformulation has undergone phase separation and has crystallized to someextent. The peak at 180 ppm showed that the amount of crystallinebudesonide had increased in the formulation of the nanoparticleagglomerates. This was consistent with the shape of several otherbudesonide peaks in the spectrum.

Budesonide consists of 25 carbons (FIG. 20); however, the spectrum ofthe budesonide as received had at least 27 resolved peaks and severalpeaks that may be the result of several overlapping peaks. The extrapeaks did not seem to be due to splitting, as would be expected if therewere more than one molecule in the asymmetric unit cell.

The budesonide is a racemic mixture of both epimers that have been shownto pack differently in the crystal lattice. Therefore, spectral editingwas used in an attempt to assign the peaks in the spectrum to determineif the differences in the two epimers could be used to explain the“extra” peaks. The spectral editing experiment allowed the assignment ofcarbon type (C, CH, CH2, or CH3) to a peak, these assignments could thenbe combined with predictions to assign the peaks to specific carbonswithin the molecule. The carbon type of most of the peaks could beassigned from these experiments (FIG. 21) with the exception of a few ofthe aliphatic peaks (particularly ˜30-−40 ppm). These results were thencompared with predictions of the solution state chemical shifts from twodifferent software programs and the resulting assignments are shown inTable 17.

TABLE 17 ¹³C peak assignments for budesonide as received. Assignment AsReceived ACD/Labs* ChemBioDraw* 20 209.0 207.0 211.2 3 185.4 186.2 185.75 170.6 170.3 168.1 1 156.7 156.5 158.4 2 122.5 and 128.2 127.5 128.3 4122.1 124.2 22  104.5 and 107.3** 105.1 102.0 17 99.8 94.4 105.0 16 83.077.3 86.7 11 67.4 and 70.7 68.0 69.0 21 67.0 66.6 9 49.7 and 53.6 54.159.0 14 48.8 44.0 13 44.6 and 47.4 47.0 44.7 10 42.6 44.1 12 32.2, 33.9,34.7, 41.0 40.4 23 38.1, 39.0, and 35.3 36.5 7 40.2 33.4 32.0 6 31.932.9 15 31.9 32.7 8 31.6 32.3 34.6 19 15.6, 18.7, and 21.0 19.0 18 22.516.3 17.3 25 14.0 14.4 24 17.2 18.4 13.1 *= Software predictions ofchemical shifts in solution. **= See text for explanation.

Chemical shifts for a compound in the solution and solid state can varyby as much as 10 ppm. For this reason, some assignments can be narroweddown to a few possibilities but an exact assignment was not possiblewith this data. For example, carbons 2 and 4 can be assigned to thepeaks at 122.5 and 128.2, but it was not possible to definitivelydetermine which carbon was associated with which peak. The solutionpredictions placed carbon 22 at ˜103 ppm, while the spectral editingexperiment showed that there are two peaks at 104.5 and 107.3 ppm thatcan be assigned to CH carbons. Additionally, carbon 22 is the chiralcenter of the epimers. Based on these observations and the fact that thetwo epimers have been shown to pack in different conformations, bothpeaks were assigned to carbon 22 with the interpretation that each peakrepresents one of the epimers. It is important to note that thisinterpretation assumes the budesonide is pure, which we have notconfirmed. Additionally, there is always the possibility that these aredifferent polymorphs; however, reports of polymorphism in budesonidehave not been identified.

The process of agglomerating nanoparticles was evaluated to determinetheir yield. The results (Table 15) have shown that the process wasefficient providing a high yield (˜90-95%) and minimum batchvariability. The loading efficiency of the drug in the preparednanoparticle agglomerates was found to be between 85-95% (Table 16),thus demonstrating minimal loss of drug during formation.

The flow characteristics of the selected nanoparticle agglomerates werealso determined (Table 18). Angle of repose, Hausner ratio and Carr'sindex are considered to be indirect methods for quantifying powderflowability. Budesonide nanoparticle agglomerates generally exhibitedsimilar bulk densities and lower tapped densities than that of the stockdrug powder. Nanoparticle agglomerates also demonstrated improved flowproperties. This may be attributed to the reduced density of thenanoparticle agglomerates. In addition, L-leucine has been reported toreduce surface energy in dry powders and may improve flowability in thiscase. Formula F3 showed slightly better flowability compared to theothers according to the Carr's index; however, all nanoparticleagglomerate powders possessed acceptable flowability.

TABLE 18 Flowability characteristics of budesonide nanoparticleagglomerates (values = average ± standard deviation). Angle of repose(θ) Tapped Carr's Formula (flow- Bulk density density index Hausner No.ability) (g/cm3) (g/cm3) (C_(i) %) ratio F1^(a) 37.1 ± 2.6 0.34 ± 0.1 0.42 ± 0.03 21.8 ± 0.5 1.2 ± 0.03 F2^(b) 39.1 ± 1.1 0.31 ± 0.1 0.40 ±0.1 23.6 ± 0.6 1.3 ± 0.03 F3^(c) 37.8 ± 2.1 0.29 ± 0.1 0.38 ± 0.1 18.6 ±0.4 1.2 ± 0.1  Stock 43.5 ± 0.7  0.30 ± 0.04 0.47 ± 0.1 35.4 ± 2.1 1.5 ±0.1  budes- onide ^(a)F1 = 0.1:0.02; Bud:Lec ^(b)F2 = 0.1:0.02:0.01;Bud:CA:PL ^(c)F3 = 0.2:0.04:0.02; Bud:CA:PL

Budesonide Nanoparticle and Nanoparticle Agglomerates Showed ImprovedDissolution Rates

A dissolution study of budesonide was conducted for the preparednanoparticles and nanoparticle agglomerates and compared to theunprocessed drug. The cumulative percentage of drug dissolved after 8hours (Q8 h) was found to be slower than that of the nanoparticles andfaster than that of the stock budesonide (Table 15). This finding wasthe expected result of increasing the surface area by decreasing theparticle size. F2 and F3 nanoparticle and nanoparticle agglomerateformulations showed faster drug dissolution than F1 which may be due tothe incorporation of the hydrophilic surfactant, PL (FIG. 22). Inaddition, increasing the concentration of this surfactant (F3) led toeven faster dissolution. Linear regression analysis of the dissolutiondata concluded that the drug was released by the Higuchi diffusionmechanism in all cases. A two-way Analysis of Variance (ANOVA) wasperformed to determine the significance of differences in dissolutionkinetics. Significant differences (α=0.05) existed betweennanoparticles, nanoparticle agglomerates and stock budesonide. Nosignificant differences existed between different nanoparticles ornanoparticle agglomerate formulations. A significant improvement(P<0.05) in the dissolution behavior of the nanoparticles andnanoparticle agglomerates was also observed when these were individuallycompared to the stock budesonide.

Formulation Components Exhibited Minimal Cytotoxicity

The cytotoxicity of the different budesonide formulations were comparedto stock budesonide, lecithin, leucine, physical mixtures of F1components and blank F1 nanoparticle agglomerates (FIG. 23). Stockbudesonide, excipients and physical mixtures of F1 components up to 5mg/mL did not show any significant cytotoxicity in A549 cells at the endof 12 hours. Blank F1 nanoparticle agglomerates did induce a very lowlevel of cytotoxicity where the IC50 was found to be 0.97 mg/mL.Additionally, F1 nanoparticles and nanoparticle agglomerates alsoinduced very low level of cytotoxicity with IC50 values equal to 1.67mg/mL and 1.91 mg/mL, respectively. The IC50 values occurred at higherconcentrations than the maximum daily dose of inhaled budesonidecurrently prescribed. These results may suggest that lecithin wasresponsible for the cytotoxic effect.

Many current techniques to generate dry powder aerosols have a majordisadvantage of poor control over the particle shape, size and sizedistribution. In addition, many pulmonary formulations may benefit fromthe improved bioavailability and rapid onset of action that may beachieved by using drug nanoparticles. In this work, budesonidenanosuspensions were successfully prepared yielding nanoparticles in therange of ˜160-230 nm. This was accomplished by using surfactants provento be safe for human use such as lecithin. Nanosuspensions wereagglomerated using L-leucine and the resulting nanoparticle agglomerateswere analyzed. Nanoparticle agglomerates were efficiently aerosolizedand offered a high fine particle fraction suitable for accessing theperipheral lung. Nanoparticle agglomerates also exhibited significantlyfaster budesonide dissolution when compared to the stock powder. Inconclusion, budesonide nanoparticle agglomerates demonstrated adesirable microstructure for efficient lung deposition and nanostructurefor rapid dissolution of poorly water soluble drugs.

Example 10 Application of Nifedipine Nanoparticles

Nifedipine (NIF), stearic acid (SA), arachidonic acid (AA), sodiumchloride and calcium chloride were purchased from Sigma Chemicals Co,U.S.A. and used as received in solid form. Acetone, methanol, ethanol,and phosphate buffered salts were purchased from Fisher Scientific andused as received. Dialysis membranes (MWCO=6-8 kDa) were purchased fromFisher Scientific. DI water was used throughout the study as obtainedfrom a Millipore ultrapurification unit present on site.

Preparation of Nifedipine Nanoparticle Suspensions

Nanoparticles were prepared by the rapid mixing of ethanol withdissolved nifedipine and stearic acid into a larger aqueous volume,known as a solvent anti-solvent precipitation technique. Briefly, 10 mgof Nifedipine and 1 mg of stearic acid were completely dissolved in 1 mlof ethanol and allowed to stir overnight. Upon complete dissolution thissolution was added to 29 ml of cold deionized water under probesonication at 60% amplitude for 20 seconds. The resulting colloid wasthen immediately frozen and lyophilized, or stored in a 4° C.refrigerator until further processing into agglomerations. At this time,a small sample was taken from the solution for sizing and imaging. Allsolution vials and reaction vessels were kept covered from any lightsources, as Nifedipine exhibits considerable photosensitivity from UVand visible light spectra.

Preparation of Nanoparticle Agglomerations

Nanoparticle colloids were destabilized via a largely understoodcombination of ionic and thermodynamic force interactions to producestable agglomerations of nanoparticles. Briefly, 30 ml of thenanoparticle suspension was taken from refrigeration and solid saltcrystals were added in various amounts. Directly after addition, thesuspensions would be subject to vigorous mixing via a homogenizationprobe operating at 20,000 RPM. Three different salt species were testedfor their ability to form agglomerations: sodium chloride, calciumchloride, and magnesium sulfate. Salts were commonly added in a 1:1ratio of salt to NIF. Colloid stability was also tested under a range ofsalt molarities and agglomeration behaviors were observed under allconditions.

Nanoparticle Characterization

Nanoparticle size, polydispersity, and zeta potential were all measuredin solution directly after synthesis and prior to agglomeration using azetaPALS dynamic light scattering device. Size and polydispersity werefirst measured. Briefly, 1 ml of the solution was added to a standardcuvette and the remaining volume was filled with deionized water. Datawere collected in three runs and combined to arrive at a final size foreach solution. Measurements were taken at 90 degrees to the incidentlight source while assuming a viscosity and refractive index of purewater. After arriving at a combined size, a second cuvette was filledwith 1 ml of our colloid solution and the remaining volume was filledwith KCl. A known voltage was then applied to this solution and datawere analyzed via online software to determine the zeta potential of theparticles in solution.

Agglomerate Characterization

Agglomerated nanoparticles were studied in solution and as a dry powder.After the agglomeration event was complete, a small volume (˜3 ml) ofthe solution was analyzed using a Beckman Coulter multisizer III. Datawere collected until the output graphs showed a stable shape andparticle counts were above 100,000.

After lyophilization, dry powders of the agglomerates were firstanalyzed using an aerosizer. At this point, 5 mg of the powder was addedto the aerosizer and data were collected until the output graphs showeda stable shape and the particle counts were above 100,000. Measurementswere taken under medium shear and no regularization.

A cascade impactor was then used to collect data on powder performancein the lung. Briefly, eight filters were pre-weighed and set ontocollection plates which were housed within eight airtight stagesarranged serially and stacked on a level setting. Air was then pumpedthrough the stages at 30 liters per minute via a vacuum pump and 10 mgof sample was introduced at the top of the impactor device. The powderswere allowed to deposit amongst the stages for 20 seconds, after whichtime the air flow was stopped. Filters were then removed from the stagesand weighed a second and final time.

Finally, the powders were characterized via two simple tests: a tapdensity test, and a test for angle of repose. The tap (bulk) density wasdetermined by demarcating a small cuvette with known volumes, and theninserting a small mass of powder into the cuvette and tapping itvertically against a padded bench top 50 times. The density was testedin triplicate. The angle of repose was measured by placing a volume ofpowder on a glass slide and tilting the slide until the powder began tomove down the slide, and recording the angle between the slide and thehorizontal. This test was also performed in triplicate for each sample.

Particle Imaging

Nanoparticles, nanoclusters, and pure drug crystals were imaged via ascanning electron microscope. The samples were deposited onto micaslides in solution (or as received for the crystals) and allowed toevaporate over night. The slides were then coated with a 2 nm goldsurface using a voltage controlled gold sputtering device and subject toa vacuum chamber whereby image data were subsequently collected.

Dissolution Studies

Dissolution events for the agglomerates, nanoparticles, and pure drugwere observed and data were recorded using a Shimadzu SPD-10A UV-Visdetector set for detection at 240 nm. 45:55 (water:methanol) mixturebuffered to pH 4.5 was used as mobile phase. Flowrates in the columnwere adjusted to 2 ml/hr and all injections were made at 50 μl. Allstudies were performed via the dialysis method in triplicate and sinkconditions were maintained at a 30:1 volume ratio. Solutions wereallowed to stir at 200 rpm without heating. Samples were introduced intodialysis bags with a molecular weight cut off of 6-8 kDa.

Results

The properties of our nanoparticle samples are shown in Table 19. It ishypothesized that including stearic acid in the formulation allows thepolar lipids to migrate to the surface of the nanoparticles arrangingtheir carbon chains to the core of the particles while allowing theirhydroxyl groups to hydrogen bond with the surrounding water molecules.This behavior is observed from the greatly reduced zeta potential of theparticles as compared with the pure drug nanoparticles (data not shown).Nifedipine is a characteristically non polar molecule, so anyaccumulation of charge on the surface of the nanoparticles may beattributed to the stearic acid. This reasoning is also aided by theobservation that stearic acid is slightly more hydrophilic thannifedipine, so it likely acts as a surfactant between the nifedipine andthe surrounding water molecules and thus migrates to the surface.

TABLE 19 Nanoparticle size, polydispersity, and zeta potential for anoptimal formulation from an ethanol solution containing 1% w/vnifedipine and using stearic acid as a stabilizer in a ratio of 10:1drug:acid. Effective Diameter (nm) 732.1 Polydispersity 0.012 ZetaPotential (mV) −26.46 Drug/Acid (mg/mg) 10 Vsolvent (mL) 1 Vantisol (mL)29

TABLE 20 Flowability parameters for three samples: Pure drug asreceived, a selected sample of nifedipine/stearic acid nanoparticles,and their corresponding agglomerates. Bulk Density Tapped Density CarrHausner Sample (g/cm³) (g/cm³) Index Ratio Pure Nif 0.205 0.227 10.001.11 Nif/SA NP 0.103 0.123 16.67 1.20 Nif/SA Floc 0.073 0.097 25.00 1.33

It was observed that a main design constraint, nanoparticle size, couldnot be easily controlled by manipulating operating conditions during theformation of the colloid (data not shown). This is probably due todominating thermodynamic force interactions in the suspension. The rateof particle precipitation is strongly dependent on the relativesolubilities of the drug in both phases (water and ethanol), and thiseffect dominates other potential factors in particle formation such asmixing energy and time. As long as there is sufficient mixing of thesetwo solvents, which can be achieved via ultrasonication at low tomoderate amplitudes, the nucleation and growth kinetics will be able toproceed as governed by the unique interfacial force balances within thesystem.

Size distributions for a sample of nanoparticle agglomerations are shownin FIG. 24. This data reveals the particle size distribution from asample of nanoparticles (geometric diameter: 421.7+/−26.2 nm, zetapotential: −32.16+/−3.75) before and after homogenization at 25,000 RPMfor 30 seconds. The data were collected in solution, and as such it isnot ideal data for studying the powder characteristics of theagglomerates. However, it is important at this stage in the synthesis toverify agglomeration, as it is well known that particles can agglomerateupon lyophilization and we wanted to verify that this is not the case inour experiments. The samples reveal a fairly monodisperse distributionof sizes between about 2 and 20 microns, with an average diameter ofabout 10 microns. More so, the data reveals very stable microstructurein the agglomerates. Their distributions are barely altered afterintense homogenization, and they maintain their shape almost entirely.

To begin powder characterization, particle samples were tested on anaerosizer via time of flight measurements. Only agglomerates were usedin the aerosizer. FIG. 25 shows a typical aerodynamic size distributionas collected via this method. The theoretical mass-mean aerodynamicdiameter (d_(aero)) of the nanoparticles was determined from thegeometric particle size and tapped density using the followingrelationship:

$d_{aero} = {\frac{d_{geo}}{\gamma}\sqrt{\rho/\rho_{a}}}$

Where d_(geo)=geometric diameter, γ=shape factor (for a sphericalparticle, γ=1; for aerodynamic diameter calculations, the particles inthis study were assumed to be spherical), ρ=particle density andρ_(a)=water mass density (1 g/cm³). As one can see from the variables,if the particle density is lower than that for water, then theaerodynamic diameter will be some fraction of the geometric diameter.This is the case for our agglomerate samples. Their geometric diametersare shown to be much larger, on average, than the aerodynamic diameters.

FIG. 26 shows SEM micrographs of the nanoparticles, agglomerates andpure drug. These images further validate the data collected, thus far.The nanoparticles are shown to bear an elliptical morphology with anaverage diameter somewhere below one micron, but not as small as 100nanometers. The agglomerate images reveal a highly textured morphology,with many small and similarly shaped lumps protruding from the surface.These features are indicative of the mechanism behind particleformation, as they are probably the result of nanoparticles groupedtogether during the agglomeration step. Also, we can see a somewhatporous assembly in the imaged marked ‘C’. Finally, the pure drug isshown to bear a highly crystalline structure, and is received incrystals larger than 100 micrometers in some cases. This crystallinemorphology is not seen in any of the other images, thus indicating thepotential change in overall crystallinity.

DSC thermographs were used to further investigate the effects ofprocessing on drug crystallinity, and to verify the overall content ineach of our formulations. As we can see from FIG. 27, both stearic acidand nifedipine draw sharp endothermic troughs where they undergo amelting phenomenon upon heating. These troughs show up in all the othergraphs, however their extent and exact shape undergo changes. Firstly,it can be seen that the area between the curves and the baselinedecrease for all processed samples. This may be due to experimentalerror, as small samples (<5 mg) were used to collect data for allparticles. This was due, in part, to their very low density and thedifficulties of handling the powders within the small sampling trays.However, it may be noted that since the particles have such a largesurface area per mass, they will be much more susceptible to heating andthus will not require as much heat to induce a state transition. Thisdifference would then show up as smaller troughs in the DSC endotherms.It is also worth noticing the consistently changed shape of thenifedipine peak for the nanoparticle samples. They reveal an exothermicpeak indicative of a crystallization event, or some other energyproducing phenomena. It may be surmised that the stearic acid andnifedipine interact upon melting of the nifedipine, whereby they arrangewith respect to each other into a semi crystalline state of lower energythan just the randomly organized fluids initially present. No othertheories for these exotherms have been presented at this point.

Dissolution studies were conducted to measure the relative ease ofnifedipine dissolution from the various forms of processed drug as seenin FIG. 28. Drug was mostly dissolved from all samples within 8 hours,or the kinetics simply slowed to a considerable halt, as was the casewith the pure nifedipine crystals. We can see here that thenanoparticles released the most drug in the least amount of time. Thisis to be expected as their smaller size allows for a greater surfacearea for the dissolution event to take place. The agglomeratesoutperformed the pure drug by a considerable margin, but no samplesreleased 100% of their material into the bulk medium. This was dueprimarily to difficulties in the experimental apparatus, as nifedipinewas inclined to adhere to the air water interfaces within thedissolution membrane and also to the membrane itself. Lack of properdispersion prevented the pure drug samples from maintaining an optimalcontact with the dissolution medium, and the same holds true for theother samples though to a lesser degree.

Finally, cascade impaction studies were performed to formalize ourpowder characterization for a pharmacological setting. The cascadeimpactor is a well known instrument devised in the 1950's for simulatingaerosol performance in the human lungs. The stages are set up so thateach of them (1-7 and F) represents a deeper layer of the pulmonary bed.Our data are shown in FIG. 29. Firstly, it should be noted that stage Fis shown here as stage 8, simple to ensure a numerical ordering to thegraph. The outputs reveal different behaviors for each of the samples.The pure drug mostly deposits in the earlier stages, 1-3. These stagesrepresent the pharynx and primary bronchi and so it may be assumed thatthese powders would not enter the lungs whatsoever. The nanoparticlesshow the bulk of their deposition between stages 4-6 and these representthe secondary, and terminal bronchiolar and alveolar regions. Theagglomerates show similar deposition patterns. Indeed, these aresuitable regions for delivery of particles to the lungs and so it may beshown here that both the nanoparticle samples and their correspondingagglomerates are able to deposit efficiently to the lungs. The exactreasons for this similarity, given different processing steps, may beelusive for the time being. However, one may consider that thenanoparticles are able to agglomerate upon lyophilization and hence bearsimilar structural properties to the agglomerates. However, thissimilarity does not necessarily detract from the advantages of theagglomeration since the agglomerated particles are more likely able tobe harvested directly from solution without the expensive processingstep of lyophilization.

Stearic acid stabilized pure drug nanoparticles of nifedipine weresynthesized via ultrasonication in a pure aqueous solution. Thesecolloids were destabilized under different salt molarities and/orsalt:drug mass ratios to induce particle agglomeration and subsequentmicrostructure formation. Nifedipine changes morphology upon processingwith and without stearic acid. Nanoparticles revealed enhanceddissolution kinetics when compared to the pure drug and agglomeratedsamples. The resulting dried powders exhibited suitable flowabilitycharacteristics and size distributions for pulmonary drug delivery.

Throughout the course of preparing samples for the primary study, datawere collected to help optimize the formulation and gain understandingof the processes at work. The results of these studies are shown here,with a brief discussion concerning their significance.

TABLE 21 Nanoparticle size, polydispersity, and zeta potential undervarious operating conditions. Sample size(nm) polydispersity zeta(mV)drug/acid disp. vol. (ml) cont. vol. (ml) son. time(s) A 235.10 0.03−20.93 50.00 1.50 30.00 90.00 B 259.60 0.01 −27.38 10.00 1.50 25.0090.00 C 262.90 0.24 −30.44 0.71 5.00 50.00 60.00 D 264.30 0.51 n/a 6.005.00 50.00 120.00 E 308.80 0.26 −19.67 5.00 1.50 25.00 60.00 F 317.80n/a −46.61 0.30 5.00 50.00 60.00 G 323.30 n/a −33.64 0.60 5.00 50.0060.00 H 336.10 n/a −34.42 0.60 5.00 50.00 60.00 I 472.30 0.15 n/a 6.001.00 50.00 20.00 J 584.40 0.01 n/a 6.00 1.00 25.00 60.00 K 598.10 n/an/a 6.00 0.10 25.00 40.00 L 635.20 0.46 n/a 6.00 5.00 10.00 60.00 M653.70 0.23 n/a 6.00 0.10 50.00 60.00

TABLE 22 Particle sizes under a range of sonication amplitudes. W/O =25, D/A = 1, Vtot = 30 ml, Prepared with 0.1% nifedipine in ethanol andstearic acid as a stabilizer. Amplitude (%) Effective Diameter (nm)Polydispersity 10 626.1 0.064 20 696.5 0.271 30 947.3 0.289 40 1057.50.005 50 1049.3 0.22 60 1303.7 0.005 70 689.8 0.354

TABLE 23 Particle sizes under a range of sonication times. W/O = 60, D/A= 10, Vtot = 30 ml, prepared with 1% nifedipine in ethanol. TimeEffective Diameter (seconds) (nm) Polydispersity Zeta Potential 5 534.3+/− 28.4 0.091 (14.32) +− 0.82 10 507.1 +/− 39.0 0.154 (12.47) +/− 0.6915 553.8 +/− 16.5 0.166 (15.65) +/− 1.25 30 507.0 +/− 20.1 0.184 (17.63)+− 2.19 60 495.8 +/− 50.2 0.26 (15.61) +/− .64

The dynamics of nanoparticle and agglomerate synthesis are somewhatelucidated from the previous tables and figures. Tables 21-23 show thatnanoparticle formation does not show immediate dependence on eithersonication amplitude or sonication time. However, it should be notedthat both tests were carried out while holding all solution variablesconstant (solvent/anti-solvent volumes, drug concentration in thesolvent phase, drug/acid ratio). It may be the case that the resultscould have shown stronger sonication amplitude and time dependencies ifthe solutions were altered.

Example 11 Application of Insulin Nanoparticles

This example describes a developed dry powder Zn-insulin formulationpossessing appropriate microstructure to reach the deep lung that isprocessed without excipients (FIG. 30). Factors such as pH and insulinconcentration were shown to have an effect on seed nanoparticle size.Circular dichroism (CD) and solid-state nuclear magnetic resonance(ssNMR) were used to show that irreversible secondary structure andcrystallinity changes of the insulin did not occur as a result ofprocessing. It has been demonstrated that excipient-free, insulinnanoclusters that are suitably sized for pulmonary delivery and have ahigh dissolution velocity can be produced with minimal processing steps.

Pure insulin nanoclusters with sizes within the respirable range wereproduced from the solvent-induced agglomeration of insulinnanoparticles. Nanoparticles were produced using titration and wereshown to have a strong correlation between pH and particle size (FIG.31). Nanoclusters were then produced using ethanol to displace theaqueous solvent and induce nanoparticle agglomeration.

Materials and Methods.

Materials.

Lyophilized insulin powder from bovine pancreas (0.5% zinc content) andphosphate buffered saline premix (PBS) were purchased from Sigma (St.Louis, Mo.). All other reagents were purchased from Fisher Scientific(Pittsburgh, Pa.) and used without further purification.

Fabrication of Insulin Nanoparticles

Approximately 100 mg of insulin stock powder was dissolved in 15 mL of0.01 N HCl solution. The solution was then titrated drop-wise to a pHjust below the isoelectric point (pI) of the native protein (5.3) with0.01 N NaOH solution, at which point the solution became colloidalwithout fully precipitating. The mean geometric diameters andpolydispersities of the nanoparticle suspension were measured usingdynamic light scattering (Brookhaven Instruments Zeta PotentialAnalyzer, Holtsville, N.Y.). Nanoparticles were diluted in deionized H₂O(100×) and three, 1 minute measurements were obtained at 25° C. for eachsample. Mean size and polydisperity were determined by the method ofcumulants. The same instrument was used to determine the zeta potential(ζ) of the nanoparticles in 1 mM potassium chloride solution. Three runsof 15 cycles were acquired, and the mean zeta potential was recorded.Some samples were frozen at −80° C. and lyophilized using a Labconcobench top lyophilizer (Kansas City, Mo.) for further analysis.

A range of pH values near the pI of the native protein were determinedin which the nanoparticle colloid was preserved. Particle sizes and zetapotentials were measured for each sample. Nanoparticle samples withinthis pH range (from 4.92 to 5.09) were centrifuged at 13,000 rpm for 10minutes and the supernatant concentration of insulin was analyzed usingUV absorbance spectroscopy (Agilent 8453). All pH values were measuredin triplicate. The measured concentration was used to calculate the massof insulin in the pellet from the original insulin mass and totalvolume.

Agglomeration of Insulin Nanoclusters.

5 mL aliquots of insulin nanoparticle suspensions were added to 15 mL ofethanol and stirred for 36 hours at 300 rpm under a fume hood.Nanoparticles with diameters of approximately 200 nm were selected forthis step. The geometric diameters of the insulin nanoclusters weremeasured using a Coulter Multisizer™ 3 (Beckman Counter, Fullerton,Calif.). Samples were then frozen at −80° C. and lyophilized for furtheranalysis.

Characterization of Aerosol Properties.

The aerodynamic diameters of the lyophilized powders were determinedusing an Aerosizer LD (Amherst Process Instruments Inc.). Data werecollected over ˜70 seconds under high shear force (−3.4 kPa) using a 700μm nozzle.

Characterization of Particle Morphology.

The size and morphology of lyophilized samples were evaluated using aLEO 1550 field emission scanning electron microscope (SEM). All sampleswere sputter-coated with gold for 30 seconds prior to imaging.

Conformational Stability of Processed Insulin.

Post-processing secondary structural changes in samples were analyzed bydissolving particles in 0.01 N HCl solution and analyzing using circulardichroism spectroscopy (CD; Jasco J-810, Easton, Md.) to determineconformational differences between processed and unprocessed insulin, aswell as thermal stability differences between groups. CD spectra wereacquired in three accumulations from 260-195 nm with a scanning speed of50 nm/min and 1.0 nm resolution. Thermal stability was determined at awavelength of 210 nm from 10-80° C. with a scanning speed of 15° C./hr.Thermal stability spectra were acquired in triplicate. Insulinconcentration in prepared solutions was determined by UV absorbancespectroscopy.

Crystallinity of Processed Insulin.

NMR: Spectra were collected using a Tecmag Apollo spectrometer operatingat 300 MHz using ramped amplitude cross-polarization (RAMP), magic-anglespinning (MAS), and SPINAL-64 decoupling. Samples were packed in 4 mmo.d. Zirconia rotors using Teflon® endcaps, and spun at 8 kHz in aChemagnetics™ Triple-Resonance HXY CP/MAS NMR probe configured to run indouble-resonance mode using the H and X channels, and fitted with a 4 mmspin module from Revolution NMR. All spectra are the sum of 120,000transients collected using a 1.5 sec pulse delay, a contact time of 2ms, and a ¹H 90° pulse width of 2.3 μs. The free induction decaysconsisted of 256 points with a dwell time of 33.3 μs. The spectra wereexternally referenced to tetramethylsilane using the methyl peak of3-methylglutaric acid at 18.84 ppm.

HPLC: The crystalline insulin content of the materials was determinedusing the method in the insulin zinc suspension monograph of the 2005U.S. Pharmacopoeia National Formulary, with minor modifications.Buffered acetone TS was produced by dissolving 8.15 g of sodium acetateand 42 g of sodium chloride in 100 mL of water, to which 68 mL of 0.1 Nhydrochloric acid and 150 mL of acetone were added, the mixture was thendiluted with water to make 500 mL. Approximately 0.5 mg of insulin wasplaced in a 1.5 mL microcentrifuge tube and 33.3 pL of a 1:2 mixture ofwater and buffered acetone TS was added to the tube to extract anyamorphous insulin. The sample was immediately centrifuged at 13,000 rpmfor one minute, the supernatant was decanted, and the extraction wasrepeated. Additionally, ˜0.5 mg of insulin was placed in anothermicrocentrifuge tube to be used as a control. Both insulin samples wereeach dissolved in 33.3 pL of 0.01 N hydrochloric acid and analyzed byHPLC, with each sample being prepared in triplicate.

The HPLC was a Shimadzu system that consisted of an SCL-10A systemcontroller, LC-10AT liquid chromatography pump, SIL-10A auto injectorwith a sample cooler, and SPD-10A UV-VIS detector with instrumentcontrol and data analysis performed through CLASS-VP software. Aqueousmobile phase was prepared by dissolving 28.4 g of anhydrous sodiumsulfate in 1000 mL of water, to which 2.7 mL of phosphoric acid wasadded and the pH was adjusted to 2.3 with ethanolamine. The aqueousmobile phase was then mixed 74:26 with acetonitrile. The separation wasperformed on a 4.6×250 mm Symmetry® C18 column from Waters that wasmaintained at 40° C. Samples were maintained at 5° C. and 20 pL wereinjected for analysis, with a mobile phase flow rate of 1 mL/min and thedetector set to 215 nm. Peak areas were normalized to the mass ofinsulin used to prepare the sample and the percent crystalline insulinwas calculated with the following equation:

Dissolution of Insulin Particles.

Approximately 6 mg of each insulin particle sample was suspended in PBS(pH 7.4). The solution was placed in a 100,000 Dalton biotech gradecellulose ester dialysis tube (Spectrum Labs, Rancho Dominiguez, Calif.)and placed in PBS solution to a final volume of 45 mL. All samples wereincubated at 37° C. and shaken at 50 rpm on a shaker table. 1 mLaliquots were taken at various time points up to 8 hours from the bulksolution and replaced with 1 mL of fresh PBS. The insulin concentrationwas measured using a Coomassie Plus colorimetric protein quantificationassay (Thermo Fisher Scientific, Waltham, Mass.). A calibration curvewas used to correlate the insulin concentration with the measuredabsorbance, with insulin concentrations ranging between 1 and 25 μg/mLused as the standard. Dissolved mass was calculated from the measuredconcentration, and was then normalized to the total loaded mass todetermine the percent dissolved. All experiments were performed intriplicate. Analysis of variance (ANOVA) was used to determinestatistically significant differences between groups (p<0.05).Comparisons among groups were done using a Fisher's F-test.

Estimation of Bulk Powder Density.

The bulk density of the dry powder was estimated using a micro-tap testapproach, as defined in the U.S. Pharmacopoeia National Formulary, withslight modifications. Briefly, dry powder samples (unprocessed insulin,nanoparticles, and nanoclusters) were added to pre-weighedmicrocentrifuge tubes, and the tubes were weighed again to determine themass of powder. The tubes were then tapped thirty times on the lab benchto compress the powder. The volume of the powder was approximated bycomparing the height of the compressed powder to that of a volume ofwater in an identical pre-weighed microcentrifuge tube. The tubecontaining the water was then weighed to determine the volume of water(assuming a density of 1 g/mL). The powder density was calculated bydividing the mass of powder by the volume of water. All samples wereanalyzed in triplicate. Analysis of variance (ANOVA) was used todetermine statistically significant differences between groups (p<0.05).Comparisons among groups were done using a Fisher's F-test.

Results

Characterization of Insulin Nanoparticles.

Zn insulin nanoparticles were created by titrating dissolved insulin tothe pl of the native protein, which resulted in a colloidal suspensionof nanoparticles. Particle sizes and zeta potentials were analyzed overa pH range of 4.92 to 5.09, and ranged from 292.5 nm to 592.1 nm (Table24). Zeta potentials ranged from 10.86 mV to 18.89 mV. Neither particlesizes nor zeta potentials correlated strongly with the pH of thesolution.

TABLE 24 Characteristics of nanoparticles at various pH values. pHDiameter (nm) Polydisperity ζ-Potential (mV) 4.92 292.5 ± 42   0.384 ±0.015 10.86 ± 2.44 4.94 535.5 ± 60.1 0.364 ± 0.008 18.89 ± 1.27 4.97345.1 ± 15.5 0.342 ± 0.02   15.6 ± 1.05 4.98 439.8 ± 57.6 0.357 ± 0.02517.95 ± 1.27 5.09 592.1 ± 61.8 0.349 ± 0.012 17.62 ± 0.27

The mass fraction of insulin remaining in solution after nanoparticleprecipitation was determined using LTV absorbance spectroscopy. Thesevalues were used to determine the mass fraction of total insulincontained in the nanoparticles. The results suggest a positivecorrelation between the particle size and the total mass of the insulinnanoparticles in suspension.

Characterization of Insulin Nanoclusters.

Insulin nanoclusters were produced from insulin nanoparticle suspensionsthrough solvent displacement. This was achieved by adding aliquots ofinsulin nanoparticle suspension to ethanol and stirring overnight. Thegeometric diameter of the insulin nanoclusters was determined to be3.408±1.35 μm. No correlation was determined to exist between insulinnanoparticle size and nanocluster size (FIG. 32). SEM imaging revealeddifferences in the morphology of the unprocessed insulin and the insulinnanoclusters (FIG. 33). The unprocessed insulin agglomerates appear tohave a more regular structure, while the nanoclusters have more of aleaf-like morphology. This leaf-like shape could aid in theaerosolizability of the insulin nanoclusters, and would suggest a shapefactor, γ, of less than 1.

Aerosol Properties of Insulin Particles.

The aerodynamic diameters of the unprocessed insulin powder, lyophilizedinsulin nanoparticles, and lyophilized insulin nanoclusters weremeasured with an Aerosizer LD and are shown in Table 25. The smalleraerodynamic diameter of the insulin nanoclusters compared to thegeometric diameter of the nanoclusters was expected because of the lowerdensity of the insulin nanoclusters (FIG. 34).

TABLE 25 Particle sizes Sample d_(geo) (μm) d_(aero) (μm) UnprocessedN/A 4.117 ± 1.852 Nanoparticles N/A 3.649 ± 2.069 Nanoclusters 3.408 ±1.35  2.32 ± 1.974

Conformational Stability of Processed Insulin.

Circular dichroism (CD) was employed to analyze the secondary structureand thermal stability of processed insulin powders. Isothermal scans ofdissolved, unprocessed insulin powder, dissolved nanoparticles, anddissolved nanoclusters reveal near-identical spectra with minima at 210nm, suggesting that any changes in secondary structure that might occurduring processing were reversible upon dissolution (FIG. 35). Thisoverlap was also reflected in the thermal stability CD scans, which showa slight change in molar ellipticity from 10-80° C. starting at about50° C. for all samples.

Crystallinity of Processed Insulin.

The crystallinity of the insulin particles was examined using ¹³C CP/MASNMR (FIG. 36). The spectra display differences in the aliphatic region(−0 to 75 ppm), although these differences are difficult to correlatewith the physical state of insulin. More obvious differences between thesamples arise in the carbonyl (−175 ppm) and aromatic (−137 ppm)regions. The peak at −137 ppm in the unprocessed insulin seems to benarrower and better resolved than peaks at −129 ppm. These same lines inthe other samples are broader, to the point where peaks at −129 ppmcannot be resolved. The peak at −175 ppm in the unprocessed insulin ismore narrow, with two very clear shoulders at −180 ppm and −173 ppm.Other samples only show one broad peak at −175 ppm.

Crystallinity of the insulin particles was also examined using thebuffered acetone method described in the U.S. Pharmacopoeia NationalFormulary. The results suggest that the unprocessed insulin particlesare between 80% and 88% crystalline; much greater than both thenanoparticles and nanoclusters, which were estimated to be between 2%and 8% crystalline, and between 17% and 24% crystalline, respectively(FIG. 37).

Dissolution of Insulin Particles.

The concentration of insulin was measured over time in PBS solution todetermine the dissolution rate of the different powders (FIG. 38). Theunprocessed insulin follows Higuchi dissolution kinetics, and thenanoparticles and nanoclusters appear to show a burst dissolutionphenomenon after 15 minutes. The dissolved masses of nanoparticles andnanoclusters were both significantly different from the dissolved massof unprocessed powder after 15 minutes (p=0.0021 and p=0.0054,respectively). The dissolved masses of neither the nanoparticles nor thenanoclusters were significantly different from the dissolved mass of theunprocessed insulin after 8 hours.

Bulk Powder Density.

The tap test method was used to determine the bulk density of theinsulin powders before and after processing. Density of the unprocessedinsulin powder was determined to be 0.48±0.08 mg/μL (FIG. 34). Thenanoparticle bulk density was determined to be 0.28±0.04 mg/μL, and thebulk density of the insulin nanoclusters was determined to be0.063±0.004 mg/μL. Analysis of variance revealed a p-value of 0.00025(p<0.05), indicating a statistically significant difference between thebulk densities of each group.

Discussion

The sizes of the nanoclusters were independent of the size of thenanoparticles used, and had a mean aerodynamic diameter that was roughlybetween 0.436 μm and 4.294 μm. This range of particle sizes is similarto other dry powder insulin formulations, such as Exubera (3.5 μm), anda formulation based on the Spiros technology (2-3 μm). Additionally,these particles were smaller than those produced using AIR technology(5-30 μm). Based on the relationship between geometric and aerodynamicdiameters, our data suggest a mean shape factor equal to 0.135 (assuminga p_(ref) of 1 mg/μL and μ_(tap)=ρ_(partide)). This value is much lessthan 1, indicating that our particles are aspherical and highlyirregular in morphology, thus making them good candidates forinhalation. This observation is further corroborated by SEM imaging(FIG. 33).

Circular dichroism was used to determine changes in the secondarystructure of insulin that might occur as a result of particleprocessing. The data suggest that there are no irreversible changes thatoccur as a result of processing, and that the thermal stability of theinsulin processed into nanoclusters and nanoparticles is neitherenhanced nor diminished (FIG. 35).

The crystallinity of the insulin particles was first examined using ¹³CCP/MAS NMR (FIG. 36). Due to their highly ordered nature, crystallinematerials will have relatively narrow lines in a ¹³C CP/MAS spectrum,while disordered or amorphous materials have relatively broad lines.Insulin consists of 51 amino acids and therefore the spectrum will bequite complicated because every amino acid will have at least an amideand a carbon, each of which will have slightly different conformationsand thus different chemical shifts. Because of this, even the ¹³C CP/MASspectrum of a crystalline protein will appear to have broad lines eventhough it of many narrow lines with slightly different chemical shifts.Therefore, it would be expected that there would be very few differencesbetween the ¹³C CP/MAS spectra of amorphous and crystalline proteins,and any differences may be subtle. The sharply resolved peaks of theunprocessed insulin would suggest that it is crystalline while all ofthe other samples appear to be amorphous, which is corroborated by thedissolution testing. However, at this time nothing can be said about thepurity of each form because there could be some crystalline insulin inthe samples that appears to be amorphous.

Crystallinity was also determined by dissolution testing, as defined bythe 2005 U.S. Pharmacopoeia National Formulary, with modifications.Buffered acetone TS was used to dissolve the amorphous insulin from eachsample, the concentration of which was then determined and used toestimate the crystallinity of the particles. The unprocessed insulin wasshown to be about 17 times more crystalline than the nanoparticles, and4 times more crystalline than the nanoclusters (FIG. 37). Thedissolution rate of both the nanoclusters and the nanoparticlesexhibited a burst effect over the first few minutes when compared to theunprocessed insulin (FIG. 38). This burst may be due to the rapiddissolution of amorphous material deposited on the surface of theparticles during processing or possibly during lyophilization. In thecase of the nanoparticles, it is probable that the large total surfacearea of the particles also plays a significant role in increasing thedissolution rate. This may be beneficial in a pulmonary insulinformulation if the desired therapeutic effect is rapid control of spikesin glucose levels. This type of formulation may be adjusted forsustained control of glucose over long periods of time, or forpostprandial glucose control.

Example 12 Formulation of Diatrozic Acid Nanoclusters

Diatrizoic acid has been validated as an effective and safe CT contrastagent for use in the lung as evidenced in both preclinical and clinicalreports in the literature. Effective delivery to the peripheral lung isimperative to access regional lymph nodes and enable the use ofdiatrizoic acid in staging lung cancer. Our original proposal reviewedthe value of this compound for this indication and demonstrated ananotechnology approach to create dry powders capable of accessing thelung periphery. Several drug compounds (e.g. budesonide) were formulatedto illustrate the fine powders enabled by this approach. Here, wedemonstrate that diatrizoic acid can indeed be formulated as a drypowder or as a nebulized suspension. Both formulations show a high fineparticle fraction, which strongly suggests that the diatrizoic acid willbe effectively delivered to the lung periphery. In addition, the lowsolubility of this compound will result in phagocytic uptake andtrafficking to the regional lymphatics to aid in diagnosis of developinglesions. The data presented here validates that our formulation ofdiatrizoic acid yields very fine particles that will access the lungperiphery whether delivered as a dry powder or, alternatively, as anebulized suspension.

Nanoparticles of diatrizoic acid. Nanosuspensions were prepared byprecipitating diatrizoic acid dissolved in ethanol into water. Theparticle size and zeta potential of the nanosuspensions were determinedby dynamic light scattering (Brookhaven, ZetaPALS). Particles as smallas 136 nm were reproducibly fabricated (Table 26). Zeta potentialmeasurements showed that the particles were positively charged. Thesolvent/non-solvent ratio of 1:10 generated the smallest diatrizoic acidnanoparticles. Zeta potential values ranged from 22-26 mV. The chargedsurface of the nanoparticles provided the potential for destabilizingthis colloid via interaction with a agglomerating agent to formnanoparticle agglomerates.

TABLE 26 Properties of diatrizoic acid nanoparticles. (values = average± standard deviation). Solvent/non- Nanoparticle Zeta-potential solventratio size (nm) (mV) 1:3 217 ± 20 26 ± 1 1:10 136 ± 36 22 ± 1 1:14 316 ±18 26 ± 1 1:29 188 ± 38 27 ± 3

Low density powders of diatrizoic acid. Low density nanoclusters arecapable of accessing the peripheral lung. Our approach is to use thenanoparticles in suspension as building blocks to create low densitynanoclusters. Diatrizoic acid nanoparticles were assembled into lowdensity nanoclusters by addition of L-leucine. L-leucine destabilizesthe colloid and causes the nanoparticles to assemble. The amount ofL-leucine added was adjusted to a drug:leucine ratio equal to 1:1. Thesize of nanoparticle agglomerates was measured using a CoulterMultisizer 3 (Beckman Coulter Inc.). The diatrizoic acid nanoparticleagglomerates were kept overnight at room temperature to evaporateethanol, frozen at −80° C., then freeze dried (Labconco, FreeZone 1).Drying lasted for 36 hours to remove all appreciable water content. Thelyophilized powder was stored at room temperature for furthercharacterization.

Low density particles were made by assembling 136 nm diatrizoic acidnanoparticles in suspension using the methods above. The particle sizewas determined from the lyophilized powder using particle time-of-flightanalysis. The particle mass mean aerodynamic diameter indentified usingthis analytical method was 1.0±0.1 μm, which is an ideal particle sizefor accessing the lung periphery.

FDA requires characterization of aerosols using cascade impaction, whichdetermines aerosol particle size; indicative of the probable depositionsite in the lung. The nanoparticle agglomerate formulation describedabove was studied using a Tisch Ambient Cascade Impactor. The study wascarried out by applying ˜20 mg powder manually into the orifice of theinstrument at an air flow rate of ˜30 L/min. Alternatively, the drypowders were suspended in water and applied into the cascade impactorthrough a nebulizer equipped with an LC Star atomizer. The suspensionwas nebulized for ˜30 minutes at the same cascade impactor flow rate.Cut-off particle aerodynamic diameters at 30 L/min for each stage of theimpactor were: pre-separator (10.00 μm), stage 0 (9.00 μm), stage 1 (5.8μm), stage 2 (4.7 μm), stage 3 (3.3 μm), stage 4 (2.1 μm), stage 5 (1.1μm), stage 6 (0.7 μm), stage 7 (0.4 μm) and filter (0 μm). Diatrizoicacid nanoparticle agglomerates deposited on each stage of the impactorwere determined by measuring the difference in weight of filters placedon the stages. The percent emitted fraction (% EF) and fine particlefractions of the total dose (FPFTD) were then calculated. In addition,the mass median aerodynamic diameter, MMAD, and geometric standarddeviation, GSD, were obtained by a linear fit of the cumulative percentless-than the particle size range by weight plotted on a probabilityscale as a function of the logarithm of the effective cut-off diameter.

Most nanoparticle agglomerates were deposited in stages 5-7 of thecascade impactor which was indicative of efficient aerosolization and ahigh fine particle fraction capable of reaching the peripheral lung.Notably, using the nebulizer for delivering the resuspended dry powderachieved a higher total mass deposition (FIG. 39). The fine particlefraction of the total dose (FPFTD) was calculated as the percentage ofaerosolized particles that reached the lower seven stages of theimpactor (corresponding to aerodynamic diameters below 5.8 μm), or thelower five stages (corresponding to aerodynamic diameters below 3.3 μm):Cascade impaction data suggested that the anticipated total lungdeposition (i.e. FPFTD<5.8 μm) was about 93-96% and deep lung deposition(i.e. FPFTD<3.3 μm) was ˜80% for diatrizoic acid aerosol formulations.

The aerosolization efficacy was represented by the percent emittedfraction (% EF). The high emitted fraction of nanoparticle agglomeratesobtained at the tested flow rate (˜76-88%) suggested efficientaerosolization of the powder (Table 27). The mass-mean geometric size ofnanoparticle agglomerates was ˜1.5 μm with a GSD of ˜2.2 μm (Table 27).Importantly, the process to agglomerate nanoparticles consistentlyachieved a high yield (˜86%), which indicated efficient processing withminimum batch variability.

TABLE 27 Cascade impaction results of lyophilized diatrizoic acidnanoparticle agglomerates (values = average ± standard deviation).Formulations Characteristics of the Diatrizoic Diatrizoic lyophilizednanoparticle acid acid agglomerates Suspension Dry powder (suspen.)(powder) At flow % EF^(e) 88 ± 1  76 ± 4 75 ± 5 70 ± 1   rate % <5.7 96± 0.3 93 ± 1 33 ± 6 21 ± 2   of ~30 FPF^(f) <3.3 81 ± 0.2 82 ± 1 10 ± 74 ± 2  L//min MMAD^(g)  1.6 ± 0.02  1.4 ± 0.04  7 ± 1 9 ± 0.1 GSD^(h)2.2 ± 0.2   2.3 ± 0.1   2 ± 0.1 2 ± 0.1 ^(e)% EF: Percent emittedfraction. ^(f)FPF: Fine particle fraction. ^(g)MMAD: Mass medianaerodynamic diameter obtained from cascade impactor. ^(h)GSD: Geometricstandard deviation

Example 13 Formulation of Moxifloxacin Nanoclusters

Various concentrations of moxifloxacin suspensions in acetone wereprepared under ultrasonication operating with an amplitude of 48% orunder homogenization at 25,000 rpm for 15 or 30 minutes, as shown inTable 28. The particle size was measured using a dynamic lightscattering instrument (Table 28).

A concentrated solution of L-leucine was then added to the selectednanosuspension while homogenizing it at 25,000 rpm for 30 s toagglomerate and form moxifloxacin nanoparticle agglomerates. The amountof L-leucine added was adjusted to a drug:leucine ratio ranging from1:0.1 to 1:1. The agglomerated suspension was allowed to stand for 30min. A Coulter Multisizer 3 (Beckman Coulter Inc.) equipped with a100-μm aperture was used to determine the geometric size distribution ofthe nanoparticle agglomerate suspensions. In addition, a puremoxifloxacin nanosuspension was kept without leucine. All samples weresnap-frozen and lyophilized to remove final traces of acetone and form aporous dry powder.

Aerodynamic size distribution of the lyophilized powder was obtained byanalyzing the time of flight measurements from an Aerosizer LD (AmherstInstruments) equipped with a 700 μm and operating 4 psi. The sizedistribution of the moxifloxacin nanoparticle suspension is shown inFIG. 40.

TABLE 28 Composition and characterization of the prepared moxifloxacinnanoparticles (values = average ± standard deviation). Conc. Formula (mgTime Nanoparticle No. %) Process¹ (min.) size (nm) Polydispersity M1 20H 15 26,493.9 ± 14   0.61 ± 1   M2 20 H 30 366.9 ± 4 0.29 ± 0.2 M3 100 S15 331.4 ± 9 0.10 ± 0.1 M4 100 S 30  418.9 ± 11 0.005 ± 0.01 M5 100 H 15175.8 ± 8 0.059 ± 0.1  M6 100 H 30  269.9 ± 12 0.05 ± 0.3 ¹S: sonicationprocess, H: homogenization process

TABLE 29 Characterization of Ciprofloxacin nanoparticle agglomerates(values = average ± standard deviation). Geometric particle sizeNanoparticle (μm) of MMAD^(c) of agglomerate Drug:L- Nanoparticlelyophilized lyophilized Formulas leucine ratio size (nm) NA^(b) NA^(b)^(a)M5-1 Pure drug 175.8 ± 8 5.08 ± 1.77 2.125 ± 1.77 M5-2 1:0.1 175.8 ±8  3.77 ± 1.146 1.795 ± 1.89 M5-3 1:0.5 175.8 ± 8 2.97 ± 0.88 1.724 ±1.88 M5-4 1:1 175.8 ± 8 3.675 ± 1.01   1.35 ± 1.93 ^(a)M5 = 25 mgmoxifloxacin/25 mg acetone; 100 mg % ^(b)NA: Nanoparticle agglomerates.^(c)MMAD: Mass median aerodynamic diameter obtained from Aerosizer.

Example 14 Formulation of Ciprofloxacin Nanoclusters

Preparation of Dry Agglomerated nanoCipro Powder

A homogenous suspension of nanoCipro was prepared by sonicating acolloidal solution of ciprofloxacin in acetone. Initial concentration ofciprofloxacin in acetone was varied between 20 mg % w/v, 40 mg % w/v and100 mg % w/v. Different amplitudes of sonication ranging from 46% to 96%were tried. The time required for sonication was also varied between 15min, 30 min, 45 min and 60 min. A solution of L-leucine was then addedto the selected nanoCipro suspensions while homogenizing it at 25,000rpm for 30 s to agglomerate and form ciprofloxacin nanoagglomerates. Theamount of L-leucine added was adjusted to a drug:leucine ratio rangingfrom 1:0.1 to 1:1. Due to poor solubility of leucine in acetone, thedesired amount of leucine was dissolved by sonication in minimal amountof water prior to injection in the nanosuspension. The agglomeratedsolution was allowed to stand for 30 min and was then left at roomtemperature to evaporate the acetone. The solution was then frozen at−80° C. and lyophilized to remove final traces of acetone and form aporous dry powder.

Particle Size Analysis

Particle size and zeta potential of nanoCipro suspensions was determinedusing a dynamic light scattering instrument (Brookhaven, ZetaPALS). In astandard cuvette, 1 mL of the nanosuspension was taken and the remainingvolume was filled with deionized water for particle size analysis and 1mM KCl for zeta potential analysis. Measurements were taken at an angleof 90° to the incident light source. A Coulter Multisizer 3 (BeckmanCoulter Inc.) equipped with a 100-μm aperture was used to determine thegeometric size distribution of the nanoparticle agglomerate suspensionsand the resuspended lyophilized powder. Approximately 2 mL of dispersedciprofloxacin nanoagglomerate solution and a pinch of the lyophilizedpowder were resuspended in ˜3 mL isotonic solution respectively for themeasurements.

Mass-Mean Aerodynamic Diameter and Tap Density Measurements.

The tap density of the dry powder was estimated using a micro-tap testapproach, as defined in the U.S. Pharmacopoeia National Formulary, withslight modifications. Briefly, lyophilized nanoagglomerate powder wasadded to pre-weighed microcentrifuge tube, and the tubes were weighedagain to determine the mass of powder. The tube was then tapped thirtytimes on the lab bench to compress the powder. The volume of the powderwas approximated by comparing the height of the compressed powder tothat of the volume of water in an identical pre-weighed microcentrifugetube. The tube containing the water was then weighed to determine thevolume of water (assuming a density of 1 g/cm³). The powder density wascalculated by dividing the mass of powder by the volume of water.

The tap density and the geometric particle size were then used tocalculate the theoretical mass-mean aerodynamic diameter. Aerodynamicsize distribution of the lyophilized powder was also obtained byanalyzing the time of flight measurements from an Aerosizer LD (AmherstInstruments) equipped with a 700 μM and operating 4 psi.

In Vitro Aerosolization Performance.

To simulate inspiration in vitro, about 5 mg of the lyophilized powderwas manually placed in the orifice of a Tisch Ambient Cascade Impactor(Tisch Environmental, Inc., Village of Cleves, Ohio, U.S.A.) operatingat an air flow rate of 30 L/min for 20 s. Powders were collected fromthe eight different stages of the impactor. The cutoff particleaerodynamic diameter for each stage was provided by the manufacturer asfollows: pre-separator (10.00 μm), stage 0 (9.00 μm), stage 1 (5.80 μm),stage 2 (4.70 μm); stage 3 (3.30 μm), stage 4 (2.10 μm), stage 5 (1.10μm), stage 6 (0.70 μm), stage 7 (0.40 μm) and the filter (0.00 μm). Massof the particles deposited on each stage was calculated by measuring thedifference in the weights of the plates placed between the stages. Then,the emitted fraction (EF) was calculated to determine the amount ofinhalable particles in the lyophilized powder using the followingequation:

The fine particle fraction of the total dose (FPFTD) which woulddetermine the amount of particles reaching the deep lung was calculatedby measuring the percentage of aerosolized particles reaching the lowerseven stages of the impactor (corresponding to aerodynamic diametersbelow 5.8 μm) as follows:

${\%\mspace{14mu}{Fine}\mspace{14mu}{particle}\mspace{14mu}{fraction}\mspace{14mu}\left( {FPF}_{TD} \right)} = {\frac{\begin{matrix}{{{Powder}\mspace{14mu}{mass}\mspace{14mu}{recovered}\mspace{14mu}{from}}\mspace{14mu}} \\{{terminal}\mspace{14mu}{stages}\mspace{14mu}{of}} \\{{the}\mspace{14mu}{impactor}}\end{matrix}}{\begin{matrix}{{Total}\mspace{14mu}{particle}\mspace{14mu}{mass}} \\{{recovered}\mspace{14mu}{in}\mspace{14mu}{the}} \\{impactor}\end{matrix}} \times 100}$

Additionally, mass median aerodynamic diameter (MMAD) and geometricstandard deviation (GSD) was calculated from a linear regression modelof cumulative mass plotted as a function of the logarithm of theeffective cut-off diameter.

Particle Imaging

Surface morphology of nanoparticle suspension, nanoagglomerates and puredrug crystals were investigated via transmission electron microscopy.Transmission electron micrographs were obtained using a JEOL 1200 EXITtransmission electron microscope. Initially, carbon-coated grids(Electron Microscopy Sciences) were coated by a droplet of thesuspensions of nanoparticles, nanoagglomerates and the drug crystalsrespectively on a flexible plastic film (Parafilm), to facilitate theadsorption of the particles onto the grid. The grid was then blottedwith a filter paper and air dried for 1 h prior to taking themicrographs.

Determination of Percent Yield and Drug Loading

The percent yield for lyophilized powder was calculated using thefollowing equation:

${\%\mspace{14mu}{Yield}} = {\frac{M_{lyophilized}}{M_{initial}} \times 100}$Where, M_(lyophlize) is the mass of powder obtained after lyophilizationof the nanoagglomerates and M_(initial) is the initial mass of solidsintroduced in acetone including the mass of pure drug crystals and theagglomerating agent added.

The drug loading efficiency was analyzed by dispersing 10 mg of thelyophilized powder in 100 mL of PBS. The solution was then sonicated ina water bath sonicator for 2 hrs and was centrifuged at 15,000 rpm for30 min. to remove any extra amount of L-leucine. The amount of drug inthe supernatant was determined spectrophotometrically at 270 nm. Drugloading was defined by the following equation:

${\%\mspace{14mu}{Loading}} = {\frac{{Recovered}\mspace{14mu}{ciprofloxacin}\mspace{14mu}{mass}}{{Total}\mspace{14mu}{mass}} \times 100}$

Drug Dissolution Studies

The in vitro dissolution of Cipro from the prepared nanoparticles andnanoparticle agglomerates was determined under sink condition andcompared with the dissolution characteristics of the drug powder asreceived. An accurately weighed amount of the lyophilized powderequivalent to 1 mg ciprofloxacin was dispersed in 10 mL phosphatebuffered saline (PBS, pH 7.4) and was suspended into a floatabledialysis membrane unit (Mw cut-off=10,000 Da). The unit was allowed tofloat in a beaker containing 150 mL PBS and the whole assembly wasstirred at a constant speed (100 rpm) using a magnetic stirrer(Barnstead, Thermolyne MIRAK™) at 37±0.5° C. Aliquots were withdrawnfrom the dialysis bag and replaced with fresh medium at predeterminedtime intervals for a total period of 8 hours. Then, the drug content wasmeasured spectrophotometrically at 270 nm. Studies were conducted intriplicate.

Results and Discussion

Fabrication of Ciprofloxacin Nanoparticles

Ciprofloxacin nanoparticles were fabricated by sonicating the drug inacetone. Different concentrations of the drug in acetone were sonicatedat various amplitudes for different times yielded mean particle sizeranging from ˜68 nm to 722 nm (Table 30). Nanosuspensions with higherconcentration of ciprofloxacin in acetone yielded very high particlesize. Increasing the amplitude resulted in a polydispersive particlesize distribution. Increasing the time required for sonication did notaffect the particle size distribution much. The desired particle size of˜68 nm was obtained when a 20 mg % w/v solution of ciprofloxacin wassonicated at amplitude of 46% for 30 min.

Agglomeration of Ciprofloxacin Nanoparticles

The mechanism to control nanoparticle agglomeration is mainly driven byleveraging the competitive processes of attraction (van der Waals force)and repulsion (electrostatic repulsive force or steric hindrance barrieror both). L-leucine, used as a agglomerating agent in these studies mayalso act as an anti-adherent material to yield a high respirablefraction of the agglomerated ciprofloxacin nanoparticles.

Agglomeration of nanoparticles resulted in the formation of agglomerateswithin the micrometer or sub-micrometer scale consisting ofclosely-packed nanoparticles. Nanoparticle agglomerates were preparedthrough the slow incorporation of L-leucine during homogenization(25,000 rpm) for 30 sec. Different amount of this agglomerating agentranging from a drug:leucine ratio of 1:0.1 to 1:1 was added to determinethe effect on particle size distribution (Table 31). The geometric sizedistribution of the prepared nanoparticle agglomerate suspensions wasmeasured in Isoton diluent using a Coulter Multisizer 3. The optimalgeometric particle size of ˜2.9 μm was obtained with a drug:leucineratio of 1:0.5. Higher or lower ratios between the drug and leucine ledto very wide size distribution or larger geometric size. The sizedistributions of resuspended lyophilized powders were slightly broaderand the average particle size was increased to ˜5.4 μm, when compared tothe nanoparticle agglomerates prior to lyophilization as shown in FIG.41. This may be due to the deposition of nanoparticles on agglomeratesduring lyophilization or to cohesion between agglomerates as a result ofdrying.

Agglomerated Ciprofloxacin Nanoparticles Yielded Desirable AerosolCharacteristics

The aerodynamic diameter (d_(aero)) is used to estimate the site ofaerosol deposition of nanoclusters within the lungs. The aerodynamicdiameter of the agglomerated nanoparticles, measured by an Aerosizer LD,was smaller than the geometric diameter indicating the low density ofnanoparticle agglomerates as shown in Table 31 and FIG. 42.

The theoretical mass-mean aerodynamic diameters (d_(aero)) of thenanoparticle agglomerates determined from the geometric particle sizeand tapped density was found to be 1.7 μm. Table 31. Tap densitymeasurements underestimate particle bulk densities since the volume ofparticles measured includes the interstitial space between theparticles. The true particle density, and therefore the aerodynamicdiameter of a given powder, is expected to be slightly larger thanreported. Ciprofloxacin nanoparticle agglomerates with d_(aero)˜1.7 areexpected to deposit primarily in the alveolar region of the lungs.

Cascade impaction studies were carried out to confirm the Aerosizerresults and theoretical MMAD calculations (FIG. 43). Most nanoparticleagglomerates were deposited in stages 4 and 5 of the cascade impactorwith a high portion of powder deposition in the lower stages. This wasindicative of efficient aerosolization and a high fine particlefraction. Conversely, drug powder as received exhibited main depositionon stages 3 & 4 with a small amount of deposition in the lower stages.The high emitted fraction of nanoparticle agglomerates obtained at thetested flow rate (˜78%) suggested efficient aerosolization of the powder(Table 32).

Cirofloxacin dry powder formulation showed an anticipated total lungdeposition (i.e. FPF_(TD)<5.8 μm)˜78% and deep lung deposition (i.e.FPF_(TD)<3.3 μm) about 52% (Table 32). However, drug powder as receiveddemonstrated lower values than that found for the dry powders. This alsowas suggestive of better aerosol performance of the nanoagglomeratescompared to the drug crystals.

The mass-mean geometric size of nanoparticle agglomerates was found tobe ˜5.4 with a GSD of ˜2.1 μm (Table 32). Typical GSD values for aerosolparticles are between 1.3-3.0. The mass-median aerodynamic diameter(MMAD) of the selected nanoparticle agglomerates, as calculated from thecascade impaction results (Table 32) was found to be 2.9 which isslightly higher than that obtained from the Aerosizer and thetheoretical MMAD calculations however this diameter indicated goodaerosolization efficiency of the prepared Cipro dry powder (Table 32).

Particle Imaging

Electron microscopy was used to study the morphology of ciprofloxacinnanoparticles and nanoparticle agglomerate formulation (C1).Transmission electron micrographs (TEM) of Ciprofloxacin nanoparticlesappeared to be very small elongated rods with smooth surface and aparticle size ˜100 nm. nanoparticles (FIG. 44A). TEM images of thenanoparticle agglomerates depicted that the nanoparticles wereagglomerated into micron sized agglomerates resembling bundles ofelongated Cipro particles as shown in FIG. 44B.

Process Yield and Drug Loading.

Cipro nanoparticle agglomerates consistently achieved a high yield(˜81%), indicating the effectiveness of the agglomeration process. Theloading efficiency of Ciprofloxacin in the prepared nanoparticleagglomerates (C1) was ˜96% (Table 31), representing minimal loss of drugduring powder preparation.

Dissolution Studies.

A dissolution study of Ciprofloxacin showed that the cumulativepercentage of drug dissolved from nanoparticle agglomerates after 8hours (Q8 h) was found to be ˜60.7% which was slower than that of thenanoparticles that achieved ˜72.8%, as shown in FIG. 45. However,nanoparticle agglomerates revealed faster dissolution compared to thestock Ciprofloxacin which exhibited ˜44.3% of 8 hours. This findingindicated that Ciprofloxacin nanoparticles and nanoparticle agglomeratesimproved the dissolution rate of the drug.

TABLE 30 Composition and characterization of the prepared Cirofloxacinnanoparticles Zeta- Formula Conc. Time Nanoparticle potential No. (mg %)Amplitude (min.) size (nm) (mV) Polydispersity C1 20 46% 30  68 ± 20 0.9 ± 0.6 0.3 ± 0.1  C2 20 72% 15 85 ± 2 1.5 ± 1 0.6 ± 0.1  C3 40 46%30  81 ± 21 0.4 ± 1 0.6 ± 0.02 C4 40 46% 60 301 ± 12 0.7 ± 4 0.3 ± 0.02C5 40 72% 30 92 ± 7 1.6 ± 3 0.4 ± 0.3  C6 40 72% 60  97 ± 21 1.3 ± 1 0.3± 0.02 C7 40 95% 30 122 ± 21 0.8 ± 2 0.4 ± 0.1  C8 100 46% 30 722 ± 680.5 ± 3 0.2 ± 0.01

TABLE 31 Characterization of Ciprofloxacin nanoparticle agglomerates(values = average ± standard deviation). Characteristics Formulation(C1^(a)) Geometric particle size (μm) of NA^(b) before 2.9 ± 1lyophilization Geometric particle size (μm) of lyophilized NA^(b) 5.4 ±1 Tab density (g/cm³)   0.1 ± 0.01 MMAD_(A) ^(c) of lyophilized NA^(b)1.2 ± 3 MMAD_(t) ^(d) of lyophilized NA^(b)   1.7 ± 0.1 Process yield oflyophilized NA^(b) 81% ± 3  Loading Efficiency of lyophilized NA^(d) 96%± 4  ^(a)C1 = 5 mg Cipro/25 mg acetone; 20 mg % ^(b)NA: Nanoparticleagglomerates. ^(c)MMAD: Mass median aerodynamic diameter obtained fromAerosizer. ^(d)MMAD: Theoretical mass mean aerodynamic diametercalculated from density measurements.

TABLE 32 Cascade impaction results of lyophilized Ciprofloxacinnanoparticle agglomerates (values = average ± standard deviation.).Formulations Characteristics of the Drug as lyophilized NA^(b) C1received At flow % EF^(c) 79 ± 4 69 ± 1   rate of % FPF^(d) <5.7 79 ± 468 ± 0.1 ~30 L//min <3.3 52 ± 1 30 ± 0.2 MMAD^(e)  2.9 ± 0.1  4.2 ± 0.03GSD^(f)  2.1 ± 0.1 2.3 ± 0.2  ^(a)C1 = 5 mg Cipro/25 mg acetone; 20 mg %^(b)NA: Nanoparticle agglomerates. ^(c)% EF: Percent emitted fraction.^(d)FPF: Fine particle fraction. ^(e)MMAD: Mass median aerodynamicdiameter obtained from cascade impactor. ^(f)GSD: Geometric standarddeviation.

Example 15 Formulation of Anti-Tuberculosis Nanoclusters

Rifampicin

This drug is freely soluble in almost all organic solvent and hascertain solubility in water ˜2.5 mg/mL which still very high percent.This solubility in water has been achieved only at 25° C., so one methodfor preparing this drug as nanoparticles is to use the precipitationtechnique where the solvent can be acetone or ethanol and theanti-solvent is very cold water. The use of an ice bath helps.

The drug as received is shown in FIG. 46. Two formulae were preparedsuccessfully by injecting 0.1% drug solution in acetone or ethanol intovery cold water in an ice bath. For acetone as solvent, particle sizewas 269.6 nm (0.008). For ethanol as solvent, particle size was 347.5 nm(0.005).

Then the samples were lyophilized directly and the dry powder wasanalyzed by SEM imaging. It was clear that most of the formulatedrifampicin dry powder, using acetone, was successfully agglomerated intonanocluster of ˜2 microns consisting of nanoparticles of ˜200 nmcompared to using ethanol and the drug as received. However, there weresome nanoparticles still unagglomerated. FIG. 47 depicts the suspensionin the acetone solvent. FIG. 48 depicts the suspension in the ethanolsolvent.

Streptomycin

By trying the solubility of streptomycin, it was found that it is freelywater soluble which indicate that it is the sulfate salt of thecompound. In addition, it was found that it is poorly soluble in acetoneand ethanol. So the process recommended is the homogenization orsonication of the compound in these organic solvent using differentconcentrations of the drug and different times. Also precipitationtechnique was carried out using water as solvent and the organic solventas anti-solvent.

Drug conc. Technique Particle size 0.1%-acetone-water ppt Very big0.1%-ethanol-water ppt Very big 0.2% in ethanol Sonic. 15 min Very big0.2% in ethanol Hom, 15 min Very big 0.2% in acetone Sonic. 15 min Verybig 0.2% in acetone Soni, 15 min Very big 0.2% in acetone Hom, 30 min511.5 nm (with some large particles)   1% in acetone Hom, 1 hrs 15 min:154.4 nm (with some large particles) 30 min: 167.9 nm (0.005)monodisperse 45 min: 238.7 nm (0.14) 1 hr: 359.8 nm-611.1 nm (with somelarge particles)

The samples after homogenization in acetone (0.2% and 1%) werelyophilized for analysis by SEM (data not shown).

Example 16 Budesonide Nanocluster Formulation

Milling with 50 Micron Grinding Media

Second preliminary small scale milling experiments had been done using50 micron grinding media to determine the suitability of this procedurefor the formulation of budesonide Nanoclusters.

However, all samples exhibited particle size ranging from 400 nm to 800nm with no degradation over the 24 hrs of the experiment. SEM imagesshowed that all most samples consisted of either separated particles of˜2 microns or agglomerates exceeding 5 microns. This may indicate theinsufficient contact between the drug and the balls or crystallizationof particles during filtration especially some samples showed particlesabsolutely similar to the drug as received (FIG. 49-63). In addition,incorporation of excipients had no great effect on the results.

Lead formulations were produced using the following conditions: 12 mg/mLbudesonide, 7 mL 50 micron media and 4 mL water (FIGS. 50) and 25 mg/mLbudesonide, 3.5 mL 50 micron media, 2 mL water (FIG. 55). These samplesshowed 3-4 micron agglomerates of small particle less than 1 micron.

FIG. 49 through FIG. 63 depict the various samples. In each figure'sdescription, “SV” refers to small vials, “LV” refers to large vials, thefirst number is the mL of water and second number is mg of media.

Milling with 200 Micron Grinding Media

Four batches were carried out using the new 200 micron YTZ media. Thefirst batch was run over 5 hours. The second batch was run over 3 hours.The third batch was run over 8 hours. The fourth batch was run over 10hours. The final lyophilized powder was analyzed by cascade impactor,SEM, and HPLC.

Milling Using 200 Micron Media for Over 3 Hours

A cascade impaction test was carried out twice using 1.5 mg of drug andthe Spinhaler for 5 sec. The analysis was done chemically using HPLC. Inthe first run, the plates were washed with 10 mL ethanol and injectedinto HPLC. In the second run, the plates were coated with silicon oilthen after the run the plates were washed with 10 mL ethanol andinjected into HPLC.

The results showed lower emitted fraction of the chemically analyzedsamples with higher MMAD compared to the sample carried out by previousprocedures (Table 33 & FIG. 64).

TABLE 33 Cascade impaction results of lyophilized budesonide mill batch2-3 hours dry powder using 200 micron media (values = average ± standarddeviation). Formulations 200 mic 200 mic 3 hrs 3 hrs 200 mic HPLCCharacteristics of the Gravimetric 3 hrs HPLC analysis with lyophilizedpowder analysis analysis coated plates At flow % EF^(b) 91 68 50 rate of% <5.7 60 36 26 ~30 L//min FPF^(c) <3.3 29 8 5 MMAD^(d) 5.1 6.8 8.8GSD^(e) 2.6 2.1 1.9 ^(a)% EF: Percent emitted fraction. ^(b)FPF: Fineparticle fraction. ^(c)MMAD: Mass median aerodynamic diameter obtainedfrom cascade impactor. ^(d)GSD: Geometric standard deviation.

Milling Using 200 Micron Media for 8 Hours.

This experiment was carried out over 8 hrs using lower concentration ofbudesonide (2.5 grams/250 mL water). Samples were collected each 30minutes after 4 hrs of starting the experiment to measure the particlesize and appearance of degradation products. Also the samples werecollected in sufficient amount and have been snap frozen directly to belyophilized for 3 days and then put in the vacuum dryer for a day.

The size started to decrease more until 7 hours followed by increaseagain. These results were confirmed by both cascade impactor and SEM.

Cascade impaction test was carried out using ˜3 mg of the powder for 5seconds only and the Spinhaler was used for the delivery of the powder.In addition, filter paper was placed each run below the final plate tominimize the passage of the powder into the tubing of the instrument.

Cascade impaction results showed decrease in the MMAD until 7 hrsfollowed by gradual increase. The MMAD for 3 hrs was 5.1 microns and for5 hours was 4.6 microns. It is obvious that 7 hours may be an optimaltime for milling the drug using 200 microns. In all samples, the emittedfraction and FPF were high indicating the improvement of the powderaerosolization compared to the previous batches prepared. However theMMAD was still slightly high, it was better that the other pure drugbatches (Table 34 & FIG. 65)

From SEM images, it was appeared that starting from 6 hours the sizedecreased until 7 hours then the particles became smaller even ˜100 nmbut agglomerated into larger agglomerates.

TABLE 34 Cascade impaction results of lyophilized budesonide mill drypowder batch 3-200 mic-8 hrs (values = average ± standard deviation).Characteristics of Formulations the lyophilized NA 4 hrs 5.5 hrs 6 hrs 7hrs 7.5 hrs 8 hrs At flow % EF^(a) 80 ± 2 99 ± 1 69 ± 2 84 ± 4 71 ± 2 78 ± 3 rate % <5.7 64 ± 1 63 ± 1  80 ± 0.1  72 ± 0.4 66 ± 1  71 ± 2 of~30 FPF^(b) <3.3 37 ± 1  42 ± 0.2 45 ± 2 48 ± 1  46 ± 0.4 45 ± 3 L//minMMAD^(c)  4.6 ± 0.7  4.1 ± 0.1 3.5 ± .4  3.2 ± 0.3 3.8 ± 0.1  3.9 ± 0.1GSD^(d) 2.8 ± 1  3.6 ± 1   3.3 ± 0.3 3.5 ± 1  3.8 ± 0.1   4 ± 0.1 ^(a)%EF: Percent emitted fraction. ^(b)FPF: Fine particle fraction. ^(c)MMAD:Mass median aerodynamic diameter obtained from cascade impactor.^(d)GSD: Geometric standard deviation.

FIG. 66 through FIG. 72 depict SEM images of the samples.

The lyophilized powder after 8 hours was analyzed by HPLC. The powderwas dissolved in acetonitrile and injected into HPLC. The resultsindicated that the degradation peak was negligible and the ratio of thedegradation peak area to the characteristic peak area was ˜0.2% howeverfor the suspension the ratio was 0.4%, Table 35 & FIG. 73. FIG. 74focuses on the degradation peak of the sample.

TABLE 35 Area under the curve (AUC) and peak height (PH) of thecharacteristic peak (CP) and degradation peak (DP) for the sample after8 hrs of millings. Peak area of Peak area of % Samples characteristicpeak degradation peak degradation 8 hrs (suspension) 1076041 5083 0.4 8hrs (powder) 2957567 5783 0.2

Milling Using 200 Micron Media for 10 Hours

This experiment was carried out over 10 hrs using budesonideconcentration of 5 grams/200 mL water. Samples were collected afterdifferent time intervals to measure the particle size and appearance ofdegradation products. Also the samples were collected after 9 and 10 hrsand snap frozen directly to lyophilized and used for further evaluationstudies. Samples after 10 hrs of milling were divided into fiveportions,

1—pure drug

2—drug/0.3% leucine

3—drug/1% leucine

4—drug/0.3% lactose

5—drug/1% lactose

The sample was lyophilized and the samples were places in vacuum dryer.Few of the powders were taken to carry out SEM. After 4 hrs, the sizestarted to increase, which suggested the formation of Nanoclusters.

TABLE 36 Particle size analysis of the samples at different timeintervals Samples Particle size (nm) 3.5 hrs 632.5 4.15 hrs  481.5   6hrs 1256.5 7.5 hrs 2893.6

Chemical Analysis Using HPLC.

Samples at different time intervals (3.5 hr, 4.15 hr, 6 hr and 10 hrs)were analyzed by HPLC. All samples were diluted with acetonitrilewithout adjusting the concentration and measured in the same day ofpreparation. The results may indicate that there was no degradation peakor non-significant peak in all samples, FIG. 75 (and FIG. 76). The ratioof the peak area of the characteristic peak to the degradation peak wasindicated in Table 37.

TABLE 37 Area under the curve (AUC) of the characteristic peak (CP) anddegradation peak (DP) for all samples. Peak area of Peak area ofSamples  characteristic peak degradation peak  % degradation   3.5 hrs3532298 — 4.15 hrs 5113106 1174 0.02   6 hrs 3664129 —   10 hrs 63934272033 0.03

SEM of batch 4—200MIC FOR 10 HRS

All images indicated that milling of budesonide powder for 10 hrs using200 micron media got very small particles (˜400 nm) agglomerated intoNanoclusters of ˜2-4 microns with the appearance of many separatednanoparticles. Addition of lactose monohydrate or leucine got the sameresults, however; increasing leucine concentration led to increase theNanocluster size, shown in FIG. 77 through FIG. 82.

Continuous Precipitation

In continuing to optimize the conditions for continuous precipitation,more trials with acetone with varying concentrations, solvent injectionrates, and non-solvent injection rates were performed. Table 38 showsall of the combinations studied.

TABLE 38 Varying conditions to create particles by continuousprecipitation. Non- Solvent Solvent Production Budes. InjectionInjection Sonication Particle Size Rate (mg/mL) (mL/min) (mL/min) (%)(nm) Polydispersity (mg/min) 2 1 10 40 526.4 ± 135.7 0.474 ± 0.025 2 5 110 40 651.1 ± 102.0 0.148 ± 0.083 5 10 1 10 40 7638.8 ± 3080.4 1.066 ±0.549 10 5 1 7.5 40 792.8 ± 94.7  0.314 ± 0.194 5 5 1 5 40 29104.4 ±10282.3 2.316 ± 0678  5 5 0.5 7.5 40 11495.0 ± 1336.3  1.647 ± 0.181 2.55 0.5 10 40 565.3 ± 61.2  0.406 ± 0.101 2.5 2 1 7.5 40 399.7 ± 46.1 0.477 ± 0.043 2 Solvent: Acetone Non-solvent: Water

Based on the particle size measurements alone, the conditions shown inbold may contain agglomerates.

The production rate of the various conditions studied is rather low withacetone. Using ethanol as the solvent can result in large particlesrather than agglomerates. The use of surfactants may reduce particlesize for conditions with increased production rates. several trials havebeen performed by adding lecithin to the solvent (Table 39). Sincelecithin has low solubility in acetone, the solvent was switched toethanol for these studies. Based on the particle size measurements, theaddition of lecithin may increase the particle size.

TABLE 39 Addition of lecithin to create particles for continuousprecipitation (Solvent: Ethanol; Non-solvent: Water). Non- SolventSolvent Production Budes. Injection Injection Lecithin Particle SizeRate (mg/mL) (mL/min) (mL/min) (%) (nm) Polydispersity (mg/min) 10 1 7.50 854.1 ± 198.9 0.154 ± 0.120 10 10 1 7.5 0.1 7801.1 ± 1776.7 0.466 ±0.027 10 10 1 7.5 0.2 2233.6 ± 1001.3 0.378 ± 0.126 10

Example 17 Budesonide Nanoclusters as MDI Formulations

A trial run for determining the feasibility of budesonide nanoclustersas Metered Dose Inhaler formulations was performed. Budesonidenanoclusters and pure budesonide (milled) were dispersed in modelpropellant HPFP (2H,3H perfluoropentane) and their dispersion stabilitywas analyzed. HPFP has similar physico-chemical properties to MDIpropellants such as HFA134a and HFA227ea. Previous literature studiesindicate that dispersion stability of therapeutic molecules is similarin both HPFP and HFA227ea.

The dispersion stability was assessed and the results are shown in FIG.83 through FIG. 87. During the study, milled budesonide began creamingat around 5 min and a clearly visible layer was seen at 1 hr. The rateof creaming was slowed when budesonide was formulated as nanoclusters.However, the reason for the steric stability of these nanoclustersdispersions in HPFP is not yet fully understood.

The study demonstrates that Budesonide nanocluster dispersions are morestable in HPFP compared to the pure milled budesonide. The stability ofnanoclusters are a function of concentration. As seen from FIG. 83through FIG. 87, 0.4 mg/ml of budesonide nanocluster are more stable to2 mg/ml. However, at both theses concentrations, complete creaming wasseen for jet milled budesonide after 3 days. The reasons for thefavorable solvation of nanocluster budesonide particles by the HPFPmolecules are not yet clear. It is possible that individual primarynanocluster nanoparticles act as steric stabilizers for the nanoclustersor it can act similarly to hollow porous particles by allowing the HPFPmolecules to enter the spaces within the nanoclusters and therebyreducing van der Waals forces. But this study indicates that thesenanoclusters could be used as modified therapeutic APIs for inhalationusing pMDIs.

Example 18 Process for Making NanoCluster Budesonide

The stock budesonide powder was suspended in water at a concentration of5 μm/300 mL. The suspension was then introduced into a MiniCer (NetzschPremier technologies, LLC) media mill containing ceramic grinding mediaof 200 micron diameter. Milling was conducted at 20° C. for 10 hours at2700 RPM. The milled suspension was subsequently frozen and dried bylyophilization.

The dried powder was tested for particle size distribution in anAnderson Cascade Impactor (ACI) using a low resistance Monodose device(Plastiape S.p.a., Italy). The results are shown below in Table 40.

TABLE 40 NanoCluster Budesonide. Characteristics of NanoCluster Batch 1Batch 2 Batch 3 % EF^(a) 85.6 83 71 ± 5 % FPF^(b) ED^(e) ≦5 76 69 55 ± 3≦3 60 54 48 ± 1 MMAD^(c) 1.9 1.9  1.5 ± 0.2 GSD^(d) 2 2.9  3.2 ± 0.05^(a)% Emitted fraction = (ED/MD) * 100 ^(b)FPF: Fine particle fraction^(c)MMAD: Mass median aerodynamic diameter (50% on cumulative massdistribution plot) ^(d)GSD: Geometric standard deviation ^(e)ED: EmittedDose

Example 19 BET Surface Area Measurement of Budesonide Nanoclusters

The surface area difference between micronized budesonide andnanocluster budesonide was determined using BET Surface and is shown inTable 41. NC Batch 25 was made using the similar conditions as Batch 4in Example 16.

TABLE 41 BET Surface area measurement Samples Surface are (m²/g) Rawmaterial 4.315 ± 0.0676 Budesonide NC Batch 25 65.54 ± 9.67 

Therefore, the present disclosure is well adapted to attain the ends andadvantages mentioned as well as those that are inherent therein. Whilenumerous changes may be made by those skilled in the art, such changesare encompassed within the spirit of this invention as illustrated, inpart, by the appended claims.

What is claimed is:
 1. A nanocluster comprising a plurality ofnanoparticles having a core of nanoparticles arranged such that thesurfaces of the nanoparticles contact adjacent nanoparticles, thenanoparticles comprise an active ingredient, and the nanocluster has amass median aerodynamic diameter of from about 0.25 μm to about 20 μm.2. The nanocluster of claim 1, wherein the mass median aerodynamicdiameter is from about 0.5 μm to about 10 μm.
 3. The nanocluster ofclaim 1, wherein the mass median aerodynamic diameter is from about 3 μmto about 20 μm.
 4. The nanocluster of claim 1, wherein the activeingredient has solubility in water of less than 200 mg/ml.
 5. Thenanocluster of claim 1, wherein the nanocluster comprises at least 50%nanoparticles by weight.
 6. The nanocluster of claim 1, wherein theactive ingredient is an anti-inflammatory compound, a bronchodilator, anantimicrobial, a vaccine, an analgesic, an anxiolytic, a hypnotic, avasodilator, a cytotoxic agent, an opiod, an anti-cancer agent, DNA,RNA, budesonide, vancomycin, moxifloxacin, mometazone, epinephrine, ¹³Clabeled urea, atropine, or diatrizoic acid.
 7. The nanocluster of claim1, wherein the nanocluster is formulated into a dry powder, asuspension, an aerosol, a spray, a tablet, or a liquid.
 8. Thenanocluster of claim 1, wherein the nanocluster is formulated togetherwith a pharmaceutically acceptable carrier.
 9. The nanocluster of claim1, wherein the nanoparticles consist essentially of the activeingredient.